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1.
BMC Plant Biol ; 21(1): 384, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34416854

RESUMO

BACKGROUND: C. sinensis is an important economic crop with fluoride over-accumulation in its leaves, which poses a serious threat to human health due to its leaf consumption as tea. Recently, our study has indicated that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, there has been a lack in CWP identification and characterization up to now. This study is aimed to characterize cell wall proteome of C. sinensis leaves and to develop more CWPs related to stress response. A strategy of combined cell wall proteomics and N-glycoproteomics was employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material. Afterwards all the proteins were subjected to UPLC-MS/MS analysis. RESULTS: A total of 501 CWPs and 195 CWPs were identified respectively by cell wall proteomics and N-glycoproteomics profiling with 118 CWPs in common. Notably, N-glycoproteomics is a feasible method for CWP identification, and it can enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional class, most of which might be involved in cell wall structure remodeling. The second largest functional class mainly encompass various proteases related to CWP turnover and maturation. Oxidoreductases represent the third largest functional class, most of which (especially Class III peroxidases) participate in defense response. As expected, identified CWPs are mainly related to plant cell wall formation and defense response. CONCLUSION: This was the first large-scale investigation of CWPs in C. sinensis through cell wall proteomics and N-glycoproteomics. Our results not only provide a database for further research on CWPs, but also an insight into cell wall formation and defense response in C. sinensis.


Assuntos
Camellia sinensis/química , Parede Celular/química , Fluoretos/análise , Glicoproteínas/análise , Folhas de Planta/química , Proteínas de Plantas/análise , China , Produtos Agrícolas/química , Proteômica
2.
Plant Physiol Biochem ; 166: 668-676, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34214777

RESUMO

Selenium (Se) is an essential element for human health and an important nutrient for plant growth. Selenite is the main form of Se available to plants in acidic soils. Previous studies have shown that phosphate transporters (PTHs) participate in selenite uptake in plants. Research on the PHT gene family is therefore vital for production of Se-rich products. Here, 23 CsPHT genes were identified in the tea (Camellia sinensis) genome and renamed based on homology with AtPHT genes in Arabidopsis thaliana. The CsPHT genes were divided into four subfamilies: PHT1, PHT3, PHT4, and PHO, containing nine, three, six, and five genes, respectively. Phylogenetic analysis indicated that fewer duplication events occurred in tea plants than in A. thaliana, rice, apple, and poplar. Genes in the same subfamily tended to share similar gene structures, conserved motifs, and potential functions. CsPHT genes were differentially expressed in various tissues and in roots under different Se levels, suggesting key roles in selenite uptake, translocation, and homeostasis. The results illuminate the contributions of CsPHT genes to selenite supply in tea plants, and lay a foundation for follow-up studies on their potential functions in this plant species.


Assuntos
Camellia sinensis , Camellia sinensis/genética , Camellia sinensis/metabolismo , Família , Regulação da Expressão Gênica de Plantas , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Selenioso , Chá
3.
Cancer Discov ; 11(5): 1100-1117, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33419761

RESUMO

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

4.
Free Radic Biol Med ; 162: 592-602, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33248265

RESUMO

The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification response. The association between Nrf2 activity and iron-related oxidative stress in neurodegenerative diseases has been studied, and Nrf2 was found to transcriptionally regulate the expression of iron transporters and ferroptosis-related factors. However, the role of Nrf2 in age-related motor dysfunction and its link to iron metabolism dysregulation in brain have not been fully elucidated. In this study, with different ages of Nrf2 knockout (KO) and wild type (WT) mice, we investigated the effects of Nrf2 deficiency on brain oxidative stress, iron metabolism and the motor coordination ability of mice. In contrast to the predicted neuroprotective role of Nrf2 in oxidative stress-related diseases, we found that Nrf2 KO remarkably improved the motor coordination of aged mice, which was associated with the reduced ROS level and decreased apoptosis of dopaminergic neurons in substantia nigra (SN) of 18-month-old Nrf2 KO mice. With high-iron and Parkinson's disease (PD) mouse models, we revealed that Nrf2 KO prevented the deposition of brain iron, particularly in SN and striatum, which may subsequently delay motor dysfunction in aged mice. The regulation of Nrf2 KO on brain iron metabolism was likely mediated by decreasing the ferroportin 1 (FPN1) level on brain microvascular endothelial cells, thus hindering the process of iron entry into the brain. Nrf2 may be a potential therapeutic target in age-related motor dysfunction diseases for its role in regulating brain iron homeostasis.


Assuntos
Células Endoteliais , Fator 2 Relacionado a NF-E2 , Envelhecimento/genética , Animais , Células Endoteliais/metabolismo , Ferro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Substância Negra/metabolismo
5.
FASEB J ; 35(2): e21174, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33200454

RESUMO

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.


Assuntos
Barreira Hematoencefálica/metabolismo , Proteínas de Transporte de Cátions/genética , Medo , Técnicas de Inativação de Genes , Hipocampo/metabolismo , Ferro/deficiência , Memória , Animais , Astrócitos/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Homeostase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo
6.
Front Genet ; 11: 598714, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33281883

RESUMO

The roots, bark, and leaves of Cinnamomum camphora are rich in essential oils, which mainly comprised monoterpenes and sesquiterpenes. Although the essential oils obtained from C. camphora have been widely used in pharmaceutical, medicinal, perfume, and food industries, the molecular mechanisms underlying terpenoid biosynthesis are poorly understood. To address this lack of knowledge, we performed transcriptome analysis to investigate the key regulatory genes involved in terpenoid biosynthesis in C. camphora. High-oil-yield trees of linalool type and low-oil-yield trees were used to assemble a de novo transcriptome of C. camphora. A total of 121,285 unigenes were assembled, and the total length, average length, N50, and GC content of unigenes were 87,869,987, 724, 1,063, and 41.1%, respectively. Comparison of the transcriptome profiles of linalool-type C. camphora with trees of low oil yield resulted in a total of 3,689 differentially expressed unigenes, among them 31 candidate genes had annotations associated with metabolism of terpenoids and polyketides, including four in the monoterpenoid biosynthesis pathway and three in the terpenoid backbone biosynthesis pathway. Collectively, this genome-wide transcriptome provides a valuable tool for future identification of genes related to essential oil biosynthesis. Additionally, the identification of a cohort of genes in the biosynthetic pathways of terpenoids provides a theoretical basis for metabolic engineering of essential oils in C. camphora.

7.
Front Pharmacol ; 11: 300, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265702

RESUMO

A safe and effective approach is needed to prevent and reduce the incidence of diabetes worldwide. The hypoglycemic efficacy of salicylic acid (salsalate, SAL), which has anti-inflammatory properties, has been empirically demonstrated in studies conducted at the Joslin Diabetes Center and elsewhere. Here, we investigated the potential role of SAL in preventing the onset of diabetes in Zucker diabetic fatty (ZDF) rats and attempted to elucidate its underlying mechanisms. ZDF and Zucker lean (ZL) rats were administered a high-fat diet with or without SAL intervention, and their relative rates of diabetes were compared. Our results showed that all rats in the placebo group developed diabetes, whereas only 10% of the SAL-treated rats presented with impaired glucose tolerance (IGT). None of the latter progressed to diabetes. Relative to the untreated rats, SAL lowered plasma glucagon and insulin while improving insulin sensitivity and ß-cell function. SAL may protect against hyperglycemia by increasing the microbial diversity, ameliorating gut dysbiosis, restoring intestinal epithelial cell connections, inhibiting endotoxin influx into the blood, and attenuating inflammation. Together, these findings suggest that SAL may be a candidate prophylactic therapy against diabetes. The protective role of SAL may be attributed to its ability to reduce intestinal inflammation and improve gut dysbiosis.

8.
MAbs ; 12(1): 1690959, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31829766

RESUMO

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.


Assuntos
Anticorpos Biespecíficos/metabolismo , Imunoglobulina G/metabolismo , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos , Reações Antígeno-Anticorpo , Linhagem Celular Tumoral , Ativação do Complemento , Complemento C1q/metabolismo , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Interferometria , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo
9.
Front Endocrinol (Lausanne) ; 11: 536213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33424763

RESUMO

Aim: Acute hyperglycemia is closely related to kidney injury. Oxidative stress activation and notable mitochondria damages were found under acute hyperglycemia treatment in our previous work. In the present study, we explored the dose-effect relationship and the pivotal role of mitophagy in acute hyperglycemia induced tubular injuries. Methods: Forty non-diabetic SD rats were randomly divided and treated with different concentrations of hyperglycemia respectively during the 6-h clamp experiment. Renal morphological and functional alterations were detected. Rat renal tubular epithelial cells were treated with different concentrations of glucose for 6 h. Markers and the regulation pathway of mitophagy were analyzed. Results: Significant tubular injuries but not glomeruli were observed under both light and electron microscope after acute hyperglycemia treatment, which manifested as enlargement of tubular epithelial cells, disarrangement of epithelial cell labyrinths and swelling of mitochondria. Urinary microalbumin, ß2-MG, CysC, NAG, GAL, and NGAL were increased significantly with the increase of blood glucose (P < 0.05). ROS was activated, mitochondrial membrane potential and LC3-II/LC3-I ratio were decreased but P62 and BNIP3L/Nix were increased in hyperglycemia groups (P < 0.05), which were reversed by AMPK activation or mTOR inhibition. Conclusion: Acute hyperglycemia causes obvious tubular morphological and functional injuries in a dose-dependent manner. Acute hyperglycemia could inhibit mitophagy through AMPK/mTOR pathway, which would aggravate mitochondria damage and renal tubular impairment.


Assuntos
Injúria Renal Aguda/etiologia , Glucose/farmacologia , Hiperglicemia/complicações , Túbulos Renais/metabolismo , Mitofagia/efeitos dos fármacos , Injúria Renal Aguda/metabolismo , Animais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hiperglicemia/metabolismo , Túbulos Renais/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitofagia/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
10.
AoB Plants ; 11(5): plz052, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31579102

RESUMO

Herkogamy is an effective way to reduce sexual interference. However, the separation of stigma and anther potentially leads to a conflict because the pollen may be placed in a location on the pollinator different from the point of stigma contact, which can reduce pollination accuracy. Floral mechanisms aiming to resolve this conflict have seldom been explored. The floral biology of protandrous Ajuga decumbens was studied to uncover how the herkogamy dilemma can be resolved. Flower anthesis was divided into male, middle, female and wilting phases. The positions of stigma and stamen were dissimilar in different flower development stages. We measured the distance of the stamen and stigma to the lower corolla lip at different floral phases, which was the pollinators' approaching way. The pollen viability, stigma receptivity, pollen removal and pollen deposition on stigma were investigated at different phases. During the male phase, the dehisced anthers were lower than the stigma, located at the pollinators' approaching way, and dispersed most pollen with high viability. As the flower developed, the anthers moved upwards, making way for pollen deposition during the female phase. Meanwhile, the stigma becomes receptive by moving into the way and consequently was deposited with sufficient pollen. The position exchange of the stamen and stigma created a dynamic herkogamy at the floral phase with different sexual functions. This floral mechanism effectively avoided sexual interference and maintained pollination accuracy. In Ajuga, the movement herkogamy might be of adaptive significance in response to the changes in the pollination environment.

11.
Sci Rep ; 9(1): 14123, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575979

RESUMO

Late embryogenesis abundant (LEA) proteins are widely known to be present in higher plants and are believed to play important functional roles in embryonic development and abiotic stress responses. However, there is a current lack of systematic analyses on the LEA protein gene family in tea plant. In this study, a total of 48 LEA genes were identified using Hidden Markov Model profiles in C. sinensis, and were classified into seven distinct groups based on their conserved domains and phylogenetic relationships. Genes in the CsLEA_2 group were found to be the most abundant. Gene expression analyses revealed that all the identified CsLEA genes were expressed in at least one tissue, and most had higher expression levels in the root or seed relative to other tested tissues. Nearly all the CsLEA genes were found to be involved in seed development, and thirty-nine might play an important role in tea seed maturation concurrent with dehydration. However, only sixteen CsLEA genes were involved in seed desiccation, and furthermore, most were suppressed. Additionally, forty-six CsLEA genes could be induced by at least one of the tested stress treatments, and they were especially sensitive to high temperature stress. Furthermore, it was found that eleven CsLEA genes were involved in tea plant in response to all tested abiotic stresses. Overall, this study provides new insights into the formation of CsLEA gene family members and improves our understanding on the potential roles of these genes in normal development processes and abiotic stress responses in tea plant, particularly during seed development and desiccation. These results are beneficial for future functional studies of CsLEA genes that will help preserve the recalcitrant tea seeds for a long time and genetically improve tea plant.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Proteínas de Plantas/genética , Sementes/genética , Estresse Fisiológico/genética , Chá/genética , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , Filogenia
12.
Int J Genomics ; 2018: 5963797, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29967765

RESUMO

The tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody perennial nonalcoholic health beverage crop. Tea seeds are categorized as recalcitrant and are sensitive to dehydration treatment. However, the molecular basis of this phenomenon has not been investigated. Thus, we analyzed the genome-wide expression profiles of three dehydration stages using RNA-Seq and digital gene expression (DGE) technologies. We performed de novo assembly and obtained a total of 91,925 nonredundant unigenes, of which 58,472 were extensively annotated. By a hierarchical clustering of differentially expressed genes (DEGs), we found that 8929 DEGs were downregulated and 5875 DEGs were upregulated during dehydration treatment. A series of genes related to ABA biosynthesis and signal transduction, transcription factor, antioxidant enzyme, LEA protein, and proline metabolism that have been reported to function in dehydration process were found to be downregulated. Additionally, the expression profiles of 12 selected genes related to tea seed dehydration treatment were confirmed by qRT-PCR analysis. To our knowledge, this is the first genome-wide study elucidating the possible molecular mechanisms of sensitivity of recalcitrant tea seeds to dehydration. The results obtained in this study contribute to the preservation of tea seeds as genetic resources and can also be used to explore the mechanism of dehydration sensitivity of other recalcitrant seeds.

13.
PLoS One ; 13(6): e0197506, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29856771

RESUMO

Tea plant (Camellia sinensis) has strong enrichment ability for selenium (Se). Selenite is the main form of Se absorbed and utilized by tea plant. However, the mechanism of selenite absorption and accumulation in tea plant is still unknown. In this study, RNA sequencing (RNA-seq) was used to perform transcriptomic analysis on the molecular mechanism of selenite absorption and accumulation in tea plant. 397.98 million high-quality reads were obtained and assembled into 168,212 unigenes, 89,605 of which were extensively annotated. There were 60,582 and 1,362 differentially expressed genes (DEGs) in roots and leaves, respectively. RNA-seq results were further validated by quantitative RT-PCR. Based on GO terms, the unigenes were mainly involved in cell, binding and metabolic process. KEGG pathway enrichment analysis showed that predominant pathways included ribosome and protein processing in endoplasmic reticulum. Further analysis revealed that sulfur metabolism, glutathione metabolism, selenocompound metabolism and plant hormone signal transduction responded to selenite in tea plant. Additionally, a large number of genes of higher expressions associated with phosphate transporters, sulfur assimilation, antioxidant enzymes, antioxidant substances and responses to ethylene and jasmonic acid were identified. Stress-related plant hormones might play a signaling role in promoting sulfate/selenite uptake and assimilation in tea plant. Moreover, some other Se accumulation mechanisms of tea plant were found. Our study provides a possibility for controlling Se accumulation in tea plant through bio-technologies and will be helpful for breeding new tea cultivars.


Assuntos
Camellia sinensis/efeitos dos fármacos , Proteínas de Plantas/genética , Selênio/toxicidade , Transcriptoma/genética , Camellia sinensis/genética , Camellia sinensis/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA , Transcriptoma/efeitos dos fármacos
14.
J Proteomics ; 176: 71-81, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29408313

RESUMO

The tea plant is a fluoride hyperaccumulator, and fluoride accumulation in its leaves is closely related to human health. To dissect molecular mechanisms underlying fluoride accumulation/detoxification, the leaves of tea seedlings exposed to different fluoride treatments for 30 days were sampled for physiological and proteomics analyses. The results showed that fluoride had no adverse effects on the growth of tea seedlings in spite of high content fluoride accumulation in their leaves. Through TMT coupled with UPLC MS/MS, 189 differentially accumulated proteins were quantified, of which 41 and 148 were localized in the cell wall and cellular compartments respectively. 41 cell wall proteins were mainly conductive to cell wall structure rearrangement, signaling modulation and the protection cells from damages; 148 cellular compartments proteins mainly contributed to diverse metabolisms reprogramming, energy reallocation and plant defense. Notably, upregulation of several proteins including GHs, smHSPs, DRT100, YLS2-like, primary amine oxidase, GDSL esterase/lipases and citrate synthase probably enhanced the defense of tea seedlings against fluoride. Collectively, our results presented a comprehensive proteomics analysis on the leaves of tea seedlings in response to fluoride, which would contribute to further deciphering of molecular mechanisms underlying fluoride accumulation/detoxification in tea plant. SIGNIFICANCE: The tea plant (Camellia sinensis) is an important economic crop with its made tea occupying up the third non-alcohol beverage in the world. Tea plant is also a fluoride hyperaccumulator with up to 98% fluoride accumulation in the leaves by initiative absorption. Due to the fact that about 40% to 90% of fluoride could be readily released into tea infusion and then absorbed by human body, overaccumulation of fluoride in tea leaves is closely related to human health. Therefore, it is very necessary to deeply dissect the mechanisms underlying fluoride accumulation/detoxification in tea plant. Previously, numerous studies were conducted to investigate fluoride specification and fluoride localization of tea plant at morphological, physiological and biochemical levels, which documented that fluoride was majorly immobilized in the cell walls and stored in the vacuoles in the form of fluoride-ligands complexes. However, the molecular mechanisms governing cell wall immobilization and vacuolar compartmentation of fluoride were still remaining unknown. Thus, a quantitative proteomics study into the leaves of tea seedlings upon exposure to fluoride was performed in current study. Our results showed that 41 and 148 of 189 differentially accumulated proteins were targeted into the cell wall and cellular compartments respectively, revealing that cell wall proteins and cellular compartments proteins played crucial roles in the response of tea seedlings to fluoride. Our results were also in good agreement with the idea that the cell wall was involved in fluoride accumulation/detoxification in tea plant. However, the functions of key interested differentially accumulated proteins need be further analyzed in follow-up work.


Assuntos
Camellia sinensis/efeitos dos fármacos , Fluoretos/farmacologia , Proteômica/métodos , Compartimento Celular , Parede Celular/química , Parede Celular/metabolismo , Fluoretos/efeitos adversos , Humanos , Folhas de Planta/metabolismo , Plântula
15.
J Biol Chem ; 293(3): 941-952, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29180448

RESUMO

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.


Assuntos
Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Anticorpos Monoclonais/metabolismo , Toxinas Bacterianas/química , Células CACO-2 , Clostridioides difficile/enzimologia , Cristalografia por Raios X , Citosol/metabolismo , Enterotoxinas/química , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Rubídio/química , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
J Biol Chem ; 292(35): 14401-14412, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28705932

RESUMO

Clostridium difficile is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.


Assuntos
Antibacterianos/metabolismo , Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimologia , Enterócitos/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferases/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Antibacterianos/química , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Neutralizantes/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Sítios de Ligação de Anticorpos , Células CACO-2 , Sequência Conservada , Cristalografia por Raios X , Enterócitos/efeitos dos fármacos , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/toxicidade , Mapeamento de Epitopos , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/toxicidade , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Sequências Repetitivas de Aminoácidos
17.
Front Plant Sci ; 8: 783, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28539934

RESUMO

A sensitive bilobed stigma is thought to assure reproduction, avoid selfing and promote outcrossing. In addition, it may also play a role in pollinator selection since only pollinators with the appropriate body size can trigger this mechanism. However, no experimental study has investigated how the sensitive stigma responds to different pollinators and its potential effects on pollination. Mazus miquelii (Phrymaceae), a plant with a bilobed stigma was studied to investigate the relationship between stigma behaviors and its multiple insect pollinators. The reaction time of stigma closure after touched, duration of temporary closure, and factors determining permanent closure of the stigma were studied when flowers were exposed to different visitors and conducted with hand pollination. Manual stimulation was also used to detect the potential differences in stigmas when touched with different degrees of external forces. Results indicated that, compared to pollinators with a small body size, larger pollinators transferred more pollen grains to the stigma, causing a rapid stigma response and resulting in a higher percentage of permanent closures. Duration of temporary closure was negatively correlated with the speed of stigma closure; a stigma that closed more rapidly reopened more slowly. Manual stimulation showed that reaction time of stigma closure was likely a response to external mechanical forces. Hand pollination treatments revealed that the permanent closure of a stigma was determined by the size of stigmatic pollen load. For large pollinators, the speedy reaction of the stigma might help to reduce pollen loss, enhance pollen germination and avoid obstructing pollen export. Stigmas showed low sensitivity when touched by inferior pollinators, which may have increased the possibility of pollen deposition by subsequent visits. Therefore, the stigma behavior in M. miquelii is likely a mechanism of pollinator selection to maximize pollination success.

18.
Ann Bot ; 119(6): 1053-1059, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28158409

RESUMO

Background and Aims: It has been suggested that the dynamics of nectar replenishment could differ for flowers after being nectar robbed or visited legitimately, but further experimental work is needed to investigate this hypothesis. This study aimed to assess the role of nectar replenishment in mediating the effects of nectar robbing on pollinator behaviour and plant reproduction. Methods: Plant-robber-pollinator interactions in an alpine plant, Salvia przewalskii , were studied. It is pollinated by long-tongued Bombus religiosus and short-tongued B. friseanus , but robbed by B. friseanus . Nectar production rates for flowers after they were either robbed or legitimately visited were compared, and three levels of nectar robbing were created to detect the effects of nectar robbing on pollinator behaviour and plant reproduction. Key Results: Nectar replenishment did not differ between flowers that had been robbed or legitimately visited. Neither fruit set nor seed set was significantly affected by nectar robbing. In addition, nectar robbing did not significantly affect visitation rate, flowers visited within a plant per foraging bout, or flower handling time of the legitimate pollinators. However, a tendency for a decrease in relative abundance of the pollinator B. religiosus with an increase of nectar robbing was found. Conclusions: Nectar robbing did not affect female reproductive success because nectar replenishment ensures that pollinators maintain their visiting activity to nectar-robbed flowers. Nectar replenishment might be a defence mechanism against nectar robbing to enhance reproductive fitness by maintaining attractiveness to pollinators. Further studies are needed to reveal the potential for interference competition among bumble bees foraging as robbers and legitimate visitors, and to investigate variation of nectar robbing in communities with different bumble bee species composition.


Assuntos
Abelhas/fisiologia , Comportamento Alimentar , Néctar de Plantas/metabolismo , Polinização , Salvia/fisiologia , Animais , China , Cadeia Alimentar , Reprodução
19.
Mol Cancer Ther ; 14(7): 1637-49, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25948294

RESUMO

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient-derived xenograft models. The antitumor activity of MEDI3622 was superior to that of EGFR/HER pathway inhibitors in the OE21 esophageal model and the COLO205 colorectal model suggesting additional activity outside of the EGFR pathway. Combination of MEDI3622 and cetuximab in the OE21 model was additive and eradicated tumors. Proteomics analysis revealed novel ADAM17 substrates that function outside of the HER pathways and may contribute toward the antitumor activity of the monoclonal antibody.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Receptores ErbB/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ADAM/imunologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab/administração & dosagem , Cetuximab/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Feminino , Células HCT116 , Células HT29 , Humanos , Camundongos Endogâmicos DBA , Camundongos Nus , Neoplasias/imunologia , Neoplasias/metabolismo , Resultado do Tratamento
20.
J Immunol Methods ; 395(1-2): 45-53, 2013 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-23831609

RESUMO

We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100µg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.


Assuntos
Anticorpos/genética , Anticorpos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Adenovírus Humanos/genética , Linhagem Celular , Antígenos Nucleares do Vírus Epstein-Barr/genética , Vetores Genéticos , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo
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