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1.
BMC Plant Biol ; 21(1): 402, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470613

RESUMO

BACKGROUND: Plant-parasitic nematodes and herbivorous insects have a significant negative impact on global crop production. A successful approach to protect crops from these pests is the in planta expression of nematotoxic or entomotoxic proteins such as crystal proteins from Bacillus thuringiensis (Bt) or plant lectins. However, the efficacy of this approach is threatened by emergence of resistance in nematode and insect populations to these proteins. To solve this problem, novel nematotoxic and entomotoxic proteins are needed. During the last two decades, several cytoplasmic lectins from mushrooms with nematicidal and insecticidal activity have been characterized. In this study, we tested the potential of Marasmius oreades agglutinin (MOA) to furnish Arabidopsis plants with resistance towards three economically important crop pests: the two plant-parasitic nematodes Heterodera schachtii and Meloidogyne incognita and the herbivorous diamondback moth Plutella xylostella. RESULTS: The expression of MOA does not affect plant growth under axenic conditions which is an essential parameter in the engineering of genetically modified crops. The transgenic Arabidopsis lines showed nearly complete resistance to H. schachtii, in that the number of female and male nematodes per cm root was reduced by 86-91 % and 43-93 % compared to WT, respectively. M. incognita proved to be less susceptible to the MOA protein in that 18-25 % and 26-35 % less galls and nematode egg masses, respectively, were observed in the transgenic lines. Larvae of the herbivorous P. xylostella foraging on MOA-expression lines showed a lower relative mass gain (22-38 %) and survival rate (15-24 %) than those feeding on WT plants. CONCLUSIONS: The results of our in planta experiments reveal a robust nematicidal and insecticidal activity of the fungal lectin MOA against important agricultural pests which may be exploited for crop protection.


Assuntos
Aglutininas/farmacologia , Arabidopsis/parasitologia , Herbivoria , Marasmius/química , Nematoides/fisiologia , Aglutininas/química , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mariposas/fisiologia , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas
2.
Biochem Genet ; 59(4): 1049-1064, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33675488

RESUMO

Treatment of acute myeloid leukemia (AML) requires new drugs as result of a rise in new cases and high disease relapse. Plant lectins with the ability to bind carbohydrates on the cell surface have the potential to treat cancer. Urtica dioica L. agglutinin (UDA) is a low weight lectin with anti-benign prostatic hyperplasia (BPH) impact. Here, we examine the impact of UDA on HL-60 cell line. Cytotoxicity and cytostatic effects were assessed in HL-60 cells treated with UDA and vincristine (positive control). The effects of the lectin on cell cycle phases and cell death mechanism were surveyed by propidium iodide (PI) staining and annexin V/PI, respectively. The activation status of the apoptosis pathway was determined by western blotting. Finally, the expression levels of 84 genes were examined by the Human cancer drug target gene PCR array kit. The results indicated that the increase in UDA concentration inhibited the proliferation of HL-60 cells as well as apoptosis induction. Cell cycle analysis showed that the number of sub G1 cells increased essentially. Experimental observations showed that UDA can induce cell apoptosis through a caspase 9-dependent pathway. The expression changes of 21 genes confirmed the apoptotic events in HL-60 cells treated with UDA. In this, we have presented the first investigation on the cytotoxic and apoptotic effects of a lectin isolated from rhizomes and roots of Urtica dioica L. on human AML cells. Generally, the results suggest that UDA may have therapeutic value for leukemia and would be studied further as a new drug for AML later on.


Assuntos
Aglutininas/farmacologia , Apoptose/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/farmacologia , Urtica dioica/química , Células HL-60 , Humanos , Leucemia Mieloide Aguda
3.
Glycoconj J ; 36(6): 473-485, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31758295

RESUMO

The emergence of multi drug resistance in non-small cell lung cancer (NSCLC) patients is a major challenge towards the efficacy of chemotherapy. Thus, there is an urgent need for the newer, better clinically targeted strategies to treat this disease. Earlier studies from our laboratory revealed the apoptotic activity of Maackia amurensis agglutinin (MAA) in human NSCLC cells. In this study, the effect of MAA on drug resistant NSCLC cells was investigated. Two Paclitaxel-resistant NSCLC sub-lines (A549/PTX100 and NCI-H460/PTX100) were developed from A549 & NCI-H460 cell lines respectively. The generation of drug resistance phenotype was confirmed by the expression of cell surface MDR-1. Both the drug resistant sub-lines showed distinct morphological alterations. MAA interacted with the cell-surface protein(s) of apparent Mr ~66 kDa and induced apoptosis in both the sub-lines through intrinsic/mitochondrial pathway, involving reduction in mitochondrial trans-membrane potential, up-regulation of Bax, unaltered/decreased expression of Bcl-XL, release of mitochondrial cytochrome c into the cytosol and activation of pro-caspases (-9&-3). Our findings highlighted the potential of this plant agglutinin to serve as an apoptosis inducing agent in drug resistant NSCLC cells.


Assuntos
Aglutininas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Maackia/química , Células A549 , Aglutininas/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspases/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína bcl-X/genética
4.
Chemosphere ; 226: 915-923, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31509921

RESUMO

Fine particulate matter ≤2.5 µm (PM2.5) is a prominent global public health risk factor that can cause respiratory infection by downregulating the amounts of antimicrobial proteins and peptides (AMPs). Both salivary agglutinin (SAG) and surfactant protein D (SPD) are important AMPs in respiratory mucosal fluid, providing protection against airway pathogen invasion and infection by inducing microbial aggregation and enhancing pathogen clearance. However, the relationship between PM2.5 and these AMPs is unclear. To better understand the relationship between PM2.5 and airway innate immune defenses, we review the respiratory antimicrobial activities of SAG and SPD, as well as the adverse effects of PM2.5 on airway innate antimicrobial defense. We speculate there exists a dual effect between PM2.5 and respiratory antimicrobial activity, which means that PM2.5 suppresses respiratory antimicrobial activity through downregulating airway AMPs, while airway AMPs accelerate PM2.5 clearance by inducing PM2.5 microbial aggregation. We propose further research on the relationship between PM2.5 and these AMPs.


Assuntos
Aglutininas/farmacologia , Antibacterianos/farmacologia , Imunidade Inata/efeitos dos fármacos , Material Particulado/efeitos adversos , Proteína D Associada a Surfactante Pulmonar/farmacologia , Sistema Respiratório/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Peptídeos Catiônicos Antimicrobianos/farmacologia , Humanos , Infecções Respiratórias/induzido quimicamente
5.
J Anim Physiol Anim Nutr (Berl) ; 103(4): 1198-1206, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30934149

RESUMO

Soya bean agglutinin (SBA) is a glycoprotein and the main anti-nutritional component in most soya bean feedstuffs. It is mainly a non-fibre carbohydrate-based protein and represents about 10% of soya bean-based anti-nutritional effects. In this study, we sought to determine the effects of N-Acetyl-D-galactosamine (GalNAc or D-GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC-J2) cells. The IPEC-J2 cells were pre-cultured with 0, 0.125 × 10-4 , 0.25 × 10-4 , 0.5 × 10-4 , 1.0 × 10-4 and 2.0 × 10-4  mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre-incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans-epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre-treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin-3 were lower in the SBA-treated groups without pre-treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin-3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre-treated groups, occludin and claudin-3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC-J2s.


Assuntos
Acetilgalactosamina/farmacologia , Aglutininas/toxicidade , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Soja/química , Suínos , Acetilgalactosamina/administração & dosagem , Aglutininas/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Células Epiteliais/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Permeabilidade , RNA/genética , RNA/metabolismo
6.
Arch Insect Biochem Physiol ; 101(1): e21543, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30854723

RESUMO

There is no study implying the effect of plant lectins on insect immune elements in both challenged and non-challenged conditions with entomopathogenic agents. Lectins may bind to immune receptors on the surface of insect hemocytes, thus inducing or even disabling common immune functions including hemocyte counts, nodulation/encapsulation, phenoloxidase activity, and synthesis of antimicrobial peptides. In the present study, effect of Polygonum persicaria L. agglutinin (PPA) on immune responses of Helicoverpa armigera Hübner was investigated by feeding artificial diet treated to the larvae. Subsequently hemocyte count and expression of some immune-related genes were considered for analyses. The two groups of larvae including control and PPA-treated (1%) were divided into four subgroups of intact, Tween-80 injected, latex-bead injected and Beauveria bassiana-injected. Except for intact larvae, the highest numbers of total and differential hemocyte counts were recorded 12 hr postinjection, however, the PPA-fed larvae showed a significantly lower hemocyte counts compared to control. The number of nodules in PPA-fed larvae was significantly lower than control, but the injected larvae of both control and PPA showed the highest nodulation 24 hr postinjection. Although the highest activity of phenoloxidase was observed 12 and 24 hr postinjection but its activity significantly decreased in PPA-fed larvae compared to control. Gene expression of antimicrobial peptides including attacin, cecropin, and peptidoglycan receptor proteins were significantly decreased in artificial diet-fed larvae containing PPA and then injected by B. bassiana spores and latex bead compared to control. These results clearly indicate adverse effects of PPA on immune responses in H. armigera.


Assuntos
Aglutininas/farmacologia , Hemócitos/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Polygonum/toxicidade , Animais , Beauveria/imunologia , Dieta/efeitos adversos , Expressão Gênica , Hemócitos/imunologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/imunologia , Larva/microbiologia , Monofenol Mono-Oxigenase/metabolismo , Mariposas/imunologia , Mariposas/microbiologia
7.
Nutr Cancer ; 71(2): 285-300, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30596280

RESUMO

Lifestyle and nutritional changes have contributed much to the somatic genetic changes which have concurrently led to an increase cancer in humans. Hence the plant-based and nutritional involvements block oncogenic transformation are in good demand. We evaluate Phloem exudates of the dietary plant, Musa acuminate pseudostem, the initial domesticated plant species with the effective lectin activity for its functional role against the tumor development and its mechanism of action. Our experimental data exhibit that Musa acuminata Lectin Protein (MALP) shows a promising cytotoxic effect against the various human cancer cell lines. Supporting this, we evaluate the in vivo anti-tumor and anti-angiogenic activity of MALP in Ehrlich Ascites Carcinoma mice model (EAC). MALP treatment resulted in tumor growth inhibition and increased the lifespan of the EAC-bearing mice without showing any side effects on normal mice, as revealed by histological parameters. Further, a significant decrease in the ascites vascular endothelial growth factor (VEGF) secretion and microvessel density supports the anti-angiogenic property of the MALP. Apoptosis-inducing activity of MALP was revealed by DNA fragmentation assay, Caspase-3 inhibitor assay and cellular morphology were studied by fluorescence staining methods. Our study delivers the real evidence that MALP with a promising an anticancer potential expressively degenerates the tumor development by affecting angiogenesis and apoptosis.


Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma de Ehrlich/irrigação sanguínea , Carcinoma de Ehrlich/tratamento farmacológico , Lectinas/farmacologia , Musa/química , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Aglutininas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Ehrlich/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Extratos Vegetais/farmacologia , Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Int J Mol Sci ; 19(2)2018 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-29462933

RESUMO

Soybean agglutinin (SBA), is a non-fiber carbohydrate related protein and a major anti-nutritional factor. Integrins, transmembrane glycoproteins, are involved in many biological processes. Although recent work suggested that integrins are involved in SBA-induced cell-cycle alterations, no comprehensive study has reported whether integrins are involved in SBA-induced cell apoptosis (SCA) in IPEC-J2. The relationship between SBA and integrins are still unclear. We aimed to elucidate the effects of SBA on IPEC-J2 cell proliferation and cell apoptosis; to study the roles of integrins in IPEC-J2 normal cell apoptosis (NCA) and SCA; and to illustrate the relationship and connection type between SBA and integrins. Thus, IPEC-J2 cells were treated with SBA at the levels of 0, 0.125, 0.25, 0.5, 1.0 or 2.0 mg/mL to determine cell proliferation and cell apoptosis. The cells were divided into control, SBA treated groups, integrin inhibitor groups, and SBA + integrin inhibitor groups to determine the integrin function in SCA. The results showed that SBA significantly (p < 0.05) lowered cell proliferation and induced cell apoptosis in IPEC-J2 (p < 0.05). Inhibition of any integrin type induced the cell apoptosis (p < 0.05) and these integrins were involved in SCA (p < 0.05). Even SBA had no physical connection with integrins, an association was detected between SBA and α-actinin-2 ACTN2 (integrin-binding protein). Additionally, SBA reduced the mRNA expression of integrins by down regulating the gene expression level of ACTN2. We concluded an evidence for the anti-nutritional mechanism of SBA by ACTN2 with integrins. Further trials are needed to prove whether ACTN2 is the only protein for connecting SBA with integrin.


Assuntos
Aglutininas/farmacologia , Apoptose/efeitos dos fármacos , Integrinas/metabolismo , Lectinas de Plantas/farmacologia , Soja/química , Actinina/genética , Actinina/metabolismo , Animais , Linhagem Celular , Integrinas/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Suínos
9.
In Vitro Cell Dev Biol Anim ; 53(8): 691-698, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28699138

RESUMO

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC50) ranging between 9 and 42 µg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.


Assuntos
Aglutininas/farmacologia , Proliferação de Células/efeitos dos fármacos , Insetos/citologia , Lectinas/farmacologia , Aglutininas/efeitos adversos , Aglutininas/química , Animais , Afídeos/citologia , Afídeos/efeitos dos fármacos , Ascomicetos/química , Linhagem Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Drosophila melanogaster/citologia , Lectinas/efeitos adversos , Lectinas/química
10.
Chin J Nat Med ; 14(11): 856-864, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27914529

RESUMO

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis (RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume (AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), and phosphorylated c-Jun NH2-terminal kinase (p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles (AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.


Assuntos
Aglutininas/farmacologia , Apoptose/efeitos dos fármacos , Arisaema/química , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Medicamentos de Ervas Chinesas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética
11.
BMC Microbiol ; 15: 237, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26502719

RESUMO

BACKGROUND: Mutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani. METHODS: The effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL. RESULTS: Knowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae. Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein. CONCLUSIONS: Taken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.


Assuntos
Aglutininas/farmacologia , Antifúngicos/farmacologia , Alho/química , Proteínas Mutantes/farmacologia , Rhizoctonia/efeitos dos fármacos , Aglutininas/isolamento & purificação , Antifúngicos/isolamento & purificação , Apoptose , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Cromatografia Líquida , Hifas/citologia , Hifas/efeitos dos fármacos , Hifas/fisiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Proteínas Mutantes/isolamento & purificação , Mapeamento de Interação de Proteínas , Espécies Reativas de Oxigênio/análise , Rhizoctonia/citologia , Rhizoctonia/fisiologia , Espectrometria de Massas em Tandem
12.
J Gen Virol ; 96(12): 3660-3666, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26407826

RESUMO

Human immunodeficiency virus type 1 (HIV-1) transmission often results from infection by a single transmitted/founder (T/F) virus. Here, we investigated the sensitivity of T/F HIV-1 envelope glycoproteins (Envs) to microbicide candidate carbohydrate-binding agents (CBAs) griffithsin (GRFT), cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA), showing that T/F Envs demonstrated different sensitivity to CBAs, with IC50 values ranging from 0.006 ± 0.0003 to >10 nM for GRFT, from 0.6 ± 0.2 to 28.9 ± 2.9 nM for CV-N and from 1.3 ± 0.2 to >500 nM for GNA. We further revealed that deglycosylation at position 295 or 448 decreased the sensitivity of T/F Env to GRFT, and at 339 to both CV-N and GNA. Mutation of all the three glcyans rendered a CBA-sensitive T/F Env largely resistant to GRFT, indicating that the sensitivity of T/F Env to GRFT is mainly determined by glycans at 295, 339 and 448. Our study identified specific T/F Env residues associated with CBA sensitivity.


Assuntos
Aglutininas/farmacologia , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/farmacologia , Galanthus/química , HIV-1/fisiologia , Lectinas de Plantas/farmacologia , Proteínas do Envelope Viral/metabolismo , Aglutininas/química , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Regulação Viral da Expressão Gênica , Proteína gp120 do Envelope de HIV , Humanos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Proteínas do Envelope Viral/genética
13.
Biochimie ; 115: 93-107, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25978938

RESUMO

Maackia amurensis agglutinin (MAA) is gaining recognition as the potential diagnostic agent for cancer. Previous studies from our laboratory have demonstrated that this lectin could interact specifically with the cells and biopsy samples of non-small cell lung cancer (NSCLC) origin but not with normal lung fibroblast cells. Moreover, this lectin was also found to induce apoptosis in NSCLC cells. Further, the biological activity of this lectin was shown to survive gastrointestinal proteolysis and inhibit malignant cell growth and tumorigenesis in mice model of melanoma thereby indicating the therapeutic potential of this lectin. Paclitaxel is one of the widely used traditional chemotherapeutic drugs for treatment of NSCLC but it exerts side-effects on normal healthy cells too. Studies have revealed that lectins have potential to act as an adjuvant chemotherapeutic agent in cancer of different origin. Thus, in the present study, an attempt was made to assess the chemo-adjuvant role of MAA in three types of NSCLC cell lines [adenocarcinoma cell line (A549), squamous cell carcinoma cell line (NCI-H520) and large cell carcinoma cell line (NCI-H460)]. We have observed that the non-cytotoxic concentration of this lectin was able to enhance the cytotoxic activity of Paclitaxel even at low dose by inducing apoptosis through intrinsic/mitochondrial pathway in all the three types of NSCLC cell lines, although the involvement of extrinsic pathway of apoptosis in case of NCI-H460 cell line could not be ruled out. Further, this lectin was also found to augment the chemo-preventive activity of this drug by arresting cells in G2-M phase of the cell cycle. Collectively, our results have suggested that Maackia amurensis agglutinin may have the potential to be used as adjuvant chemotherapeutic agent in case of NSCLC.


Assuntos
Aglutininas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Maackia/química , Paclitaxel/farmacologia , Aglutininas/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Lectinas de Plantas/metabolismo
14.
J Antimicrob Chemother ; 70(6): 1674-85, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25700718

RESUMO

OBJECTIVES: This study aimed to assess the antiviral properties of a unique lectin (NICTABA) produced by the tobacco plant, Nicotiana tabacum. METHODS: Cellular assays were used to investigate the antiviral activity of NICTABA and Urtica dioica agglutinin (UDA). Surface plasmon resonance (SPR) studies were performed to study the sugar specificity and the interactions of both lectins with the envelope glycoproteins of HIV-1. RESULTS: The N-acetyl-d-glucosamine (GlcNAc)-binding lectins exhibited broad-spectrum activity against several families of enveloped viruses including influenza A/B, Dengue virus type 2, herpes simplex virus types 1 and 2 and HIV-1/2. The IC50 of NICTABA for various HIV-1 strains, clinical isolates and HIV-2 assessed in PBMCs ranged from 5 to 30 nM. Furthermore, NICTABA inhibited syncytium formation between persistently HIV-1-infected T cells and uninfected CD4+ T lymphocytes and prevented DC-SIGN-mediated HIV-1 transmission to CD4+ target T lymphocytes. However, unlike many other antiviral carbohydrate-binding agents (CBAs) described so far, NICTABA did not block HIV-1 capture to DC-SIGN+ cells and it did not interfere with the binding of the human monoclonal antibody 2G12 to gp120. SPR studies with HIV-1 envelope glycoproteins showed that the affinity of NICTABA for gp120 and gp41 was in the low nanomolar range. The specific binding of NICTABA to gp120 could be prevented in the presence of a GlcNAc trimer, but not in the presence of mannose trimers. NICTABA displayed no antiviral activity against non-enveloped viruses. CONCLUSIONS: Since CBAs possess a high genetic barrier for the development of viral resistance and NICTABA shows a broad antiviral activity profile, this CBA may qualify as a potential antiviral candidate with a pleiotropic mode of action aimed at targeting the entry of enveloped viruses.


Assuntos
Acetilglucosamina/metabolismo , Aglutininas/farmacologia , Antivirais/farmacologia , Lectinas/farmacologia , Vírus/efeitos dos fármacos , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Aglutininas/isolamento & purificação , Antivirais/isolamento & purificação , Humanos , Concentração Inibidora 50 , Lectinas/isolamento & purificação , Ligação Proteica , Ressonância de Plasmônio de Superfície , Tabaco/química , Urtica dioica/química
15.
Glycobiology ; 25(4): 428-37, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25472443

RESUMO

The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one  Leishmania donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild type but similar in size to LPG from wild type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(ß1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% is branched trisaccharide Gal(ß1,4)[Glc(ß1,2)]Man and tetrasaccharide Gal(ß1,4)[Glc(ß1,2)Man(α1,2)]Man instead of the usual Gal(ß1,4)Man and Man(α1,2)Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a 2-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a ß-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms.


Assuntos
Aglutininas/farmacologia , Glicoesfingolipídeos/metabolismo , Leishmania donovani/efeitos dos fármacos , Ricina/farmacologia , Configuração de Carboidratos , Sequência de Carboidratos , Resistência a Medicamentos , Galactose/química , Galactose Oxidase/química , Glicoesfingolipídeos/química , Glicosilação , Leishmania donovani/metabolismo , Dados de Sequência Molecular
16.
J Insect Physiol ; 70: 94-101, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25240534

RESUMO

In the last decades lectins have received a lot of attention as potential tools in pest control. Despite substantial progress in the field not all the factors determining insecticidal potency and selectivity of these proteins have been described. Recently, three lectins, RSA (Rhizoctonia solani agglutinin), SNA-I and SNA-II (Sambucus nigra agglutinin I and II) have been shown to be toxic to aphids and caterpillars. In this project we investigated if these lectins are also toxic against larvae and a cell line of the red flour beetle, Tribolium castaneum, a model organism and important pest of stored products. Furthermore, we analyzed the stability of the lectins in the larval gut and used confocal microscopy to compare their efficiency in passing through the peritrophic matrix (PM). We observed that all three lectins were toxic against the T. castaneum cell line and their effectiveness in vitro was in decreasing order SNA-II>SNA-I>RSA with the respective EC50 being 0.1, 0.5 and 3.6 µg/ml. Larvae feeding for 16 day on diets containing 2% RSA, 2% SNA-II and 2% SNA-I weighed 0.14 ± 0.07 mg, 0.67 ± 0.44 mg and 1.89 ± 0.38 mg, corresponding to approximately 7%, 36% and 80% of control larvae, respectively. As a consequence, RSA increased the time to adult emergence by over 3-fold, SNA-II by 1.9-fold and SNA-I by 1.2-fold. RSA and SNA-II were stable in the larval gut, while SNA-I was digested and excreted with the feces. Finally, confocal microscopy confirmed that RSA passed through the PM more efficiently than SNA-II. In conclusion, our data suggest that the lectin ability to pass through the PM, governed by molecule dimensions, charge and size of PM pores, is one of the features that determine the toxicity of these insecticidal proteins.


Assuntos
Lectinas/farmacologia , Tribolium/efeitos dos fármacos , Aglutininas/farmacologia , Animais , Trato Gastrointestinal/metabolismo , Larva/efeitos dos fármacos , Controle Biológico de Vetores/métodos , Lectinas de Plantas/farmacologia , Rhizoctonia , Proteínas Inativadoras de Ribossomos/farmacologia
17.
J Antimicrob Chemother ; 69(10): 2746-58, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24970741

RESUMO

OBJECTIVES: Oscillatoria agardhii agglutinin homologue (OAAH) proteins belong to a recently discovered lectin family. The founding member OAA and a designed hybrid OAAH (OPA) recognize similar but unique carbohydrate structures of Man-9, compared with other antiviral carbohydrate-binding agents (CBAs). These two newly described CBAs were evaluated for their inactivating properties on HIV replication and transmission and for their potential as microbicides. METHODS: Various cellular assays were used to determine antiviral activity against wild-type and certain CBA-resistant HIV-1 strains: (i) free HIV virion infection in human T lymphoma cell lines and PBMCs; (ii) syncytium formation assay using persistently HIV-infected T cells and non-infected CD4+ T cells; (iii) DC-SIGN-mediated viral capture; and (iv) transmission to uninfected CD4+ T cells. OAA and OPA were also evaluated for their mitogenic properties and potential synergistic effects using other CBAs. RESULTS: OAA and OPA inhibit HIV replication, syncytium formation between HIV-1-infected and uninfected T cells, DC-SIGN-mediated HIV-1 capture and transmission to CD4+ target T cells, thereby rendering a variety of HIV-1 and HIV-2 clinical isolates non-infectious, independent of their coreceptor use. Both CBAs competitively inhibit the binding of the Manα(1-2)Man-specific 2G12 monoclonal antibody (mAb) as shown by flow cytometry and surface plasmon resonance analysis. The HIV-1 NL4.3(2G12res), NL4.3(MVNres) and IIIB(GRFTres) strains were equally inhibited as the wild-type HIV-1 strains by these CBAs. Combination studies indicate that OAA and OPA act synergistically with Hippeastrum hybrid agglutinin, 2G12 mAb and griffithsin (GRFT), with the exception of OPA/GRFT. CONCLUSIONS: OAA and OPA are unique CBAs with broad-spectrum anti-HIV activity; however, further optimization will be necessary for microbicidal application.


Assuntos
Aglutininas/farmacologia , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Lectinas/farmacologia , Oscillatoria/metabolismo , Aglutininas/metabolismo , Fármacos Anti-HIV/metabolismo , Proteínas de Bactérias/farmacologia , Linhagem Celular , Farmacorresistência Viral , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Lectinas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Testes de Sensibilidade Microbiana , Ligação Proteica
18.
Biochem Biophys Res Commun ; 447(4): 586-9, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24747075

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Fúngicas/farmacologia , Proteína X Associada a bcl-2/metabolismo , Aglutininas/metabolismo , Aglutininas/farmacologia , Aglutininas/toxicidade , Substituição de Aminoácidos , Animais , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/toxicidade , Variação Genética , Lectinas/metabolismo , Lectinas/farmacologia , Lectinas/toxicidade , Marasmius/química , Marasmius/genética , Camundongos , Células NIH 3T3 , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Ácido Nucleico/toxicidade , Inibidores da Síntese de Proteínas/farmacologia , Inibidores da Síntese de Proteínas/toxicidade
19.
PLoS One ; 8(9): e74363, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24019967

RESUMO

Pinelliapedatisecta agglutinin (PPA) has previously been used in labeling fractions of myeloid leukemia cells in our laboratory. We report here that a bacterial expressed recombinant PPA domain b tagged with soluble coxsackie and adenovirus receptor (sCAR-PPAb) preferentially recognized drug resistant cancer cells K562/ADR and H460/5Fu, as compared to their parental cell lines. Pretreatment of K562/ADR cells with sCAR-PPAb significantly enhanced phagocytosis of K562/ADR by macrophages in vivo. Meanwhile, in a K562/ADR xenograft model, intratumoral injection of sCAR-PPAb induced macrophage infiltration and phagocytosis. Furthermore, immunoprecipitation, mass spectrometry and Western blot identified the membrane target of PPA on K562/ADR as sarcolemmal membrane associated protein (SLMAP). An antibody against SLMAP significantly promoted the phagocytosis of K562/ADR by macrophages in vitro. These findings suggest that PPA not only could be developed into a novel agent that can detect drug resistant cancer cells and predict chemotherapy outcome, but also it has potential value in immunotherapy against drug resistant cancer cells through inducing the tumoricidal activity of macrophages.


Assuntos
Aglutininas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Pinellia/metabolismo , Sarcolema/metabolismo , Aglutininas/metabolismo , Animais , Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Células K562 , Macrófagos/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Ligação Proteica
20.
Placenta ; 34(9): 757-64, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23806179

RESUMO

INTRODUCTION: Activated uterine natural killer (uNK) cells are abundant in early human and mouse decidual basalis. In mice, distinct uNK cell subsets support early endothelial tip cell induction, the pruning of new vessels and initiation of spiral arterial modification. While genetic studies indicate that NK/uNK cell activation via receptors recognizing Class I MHC-derived peptides promotes human pregnancy, roles for other activation receptors expressed by NK cells, such as the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptors (NCR) are undefined in human or mouse pregnancies. METHODS: Expression of AHR and NCR1 (ortholog of human NKp46) by gestation day (gd)10.5 mouse uNK cell subsets was measured by quantitative real-time RT-PCR. Early implantation sites from mice lacking expression of either receptor were examined histologically. RESULTS: Gd10.5 uNK cell subsets, separated by reactivity to Dolichos biflorus agglutinin lectin, differed in relative transcript abundance for Ahr and Ncr1. Quantitative histology revealed that, in comparison to C57BL/6 controls, implant sites from gd10.5 Ahr(-/-) and gd6.5-12.5 UkCa:B6.Ncr1(Gfp/Gfp) mice had normal uNK cell abundance but the uNK cells were smaller than normal and unable to trigger spiral arterial remodeling. Whole mount immunohistochemistry comparisons of viable, gd6.5-8.5 Ncr1(Gfp/Gfp) and C57BL/6 implant sites revealed deficits in implant site angiogenesis and conceptus growth in Ncr1(Gfp/Gfp). DISCUSSION: In mice, activation of AHR and of NCR1 by endogenous, as yet undefined ligands, contributes to uNK cell activation/maturation and angiogenic functions during early to mid-gestation pregnancy. MHC-independent activation of uNK cells also likely makes critical contributions to human pregnancy success.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Decídua/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Receptor 1 Desencadeador da Citotoxicidade Natural/agonistas , Receptores de Hidrocarboneto Arílico/agonistas , Útero/imunologia , Aglutininas/farmacologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Decídua/citologia , Decídua/metabolismo , Implantação do Embrião/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucopoese/efeitos dos fármacos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Placentação/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Gravidez , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Útero/citologia , Útero/efeitos dos fármacos , Útero/metabolismo
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