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Purification and structural studies of a Tremella fuciformis mushroom lectin
Get PDFLectins are carbohydrate-binding proteins or glycoproteins of non-immune origine widely distributed in living organisms including animals, plants and fungi. They play a role in different biological processes mediating cellular signaling, differentiation, tissue metastasis and host-pathogen interactions. Moreover they serve as storage proteins, are fundamental during fungi and plant morphogenesis and development and take part into their defense processes [1].Thanks to their carbohydrate specific binding, some lectins are able to recognize, in a reversible way, the sugar moieties on the erythrocytes cell surface (N-acetylgalactosamine, D-galactosamine), causing a phenomena called hemagglutination. Furthermore some lectins have been found to possess antitumoral properties [2]. Specifically they recognize the Tn-antigenic determinant (Galβ1-3GalNAcα) on the malignant cells surface causing apoptosis, cytotoxicity, inhibition of tumor growth and preventing the proliferation of tumor cells. Considering the fact that this kind of residues are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells, also because the Tn-antigen is the most specific human cancer-associated structure, expressed in about 90% of the human carcinomas.For the reasons described above, during the last decades lectins have been extensively investigated for their potential therapeautical effects and biotechonological applications, especially fungal lectins which have unique carbohydrate specificities. However, altough the function and the biological properties of many lectins have been determined, their structural characterization lags behind.As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and have shown to possess anticancer, anti-inflammatory, antioxidant and neuroprotective activities. In the light of above the crude extract proteins have been checked to assess the presence of lectins [3]. To this purpose, the mushrooms dried fruiting bodies of Tremella fuciformis were homogenized and extracted in a phospate buffer at 4°C and neutral pH. The crude extract was then precipitated using a high concentration of (NH4)2SO4 and dyalised against TRIS buffer in order to remove the precipitant. A lectin was eluted from a hog gastric mucin affinity column and purified first with a DEAE-cellulose column and then with a size exclusion SEPHACRYL G-100 column. An electrophoresis gel was required to precisely define the lectin molecular weight, which is 22 kDa. The purified lectin has been used for testing several crystal screening conditions
Structural and biophysical studies on the lectin domain of GalNAc-T6 for therapeutic applications
Get PDFThe expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Galβ1–3GalNAc; T-antigen) and the Lewis antigen (sialyl-T-antigen) is a characteristic feature observed on almost all malignant epithelial cells. Therefore, there is a particular interest in their application not only as prognostic markers but also as therapeutic targets [1]. These antigens can be recognized by lectins, a group of highly specific carbohydrate-binding proteins that have been proposed as useful tools for antitumor drug-targeting [2].The three-dimensional structure of several lectins with antitumor properties has been determined in our laboratory by X-ray crystallography. N-α-acetylgalactosaminyltransferase-6 (GalNAc-T6) is an enzyme present also in humans which contains a catalytic domain and a lectin domain with a binding site for N-acetylgalactosamine (GalNAc), one of the saccharides exposed by cancer cells (Tn-antigen). Unlike other lectins with these properties, the lectin domain of GalNAc-T6 presents a structural fold found also in other human proteins, unlocking the opportunity to use protein engineering tools to design new anticancer therapeutics [3]. The three-dimensional structure of GalNAc-T6 has not been determined so far, neither has been its substrate specificity. Therefore, the production of a recombinant form containing only the lectin domain can contribute to these two critical points that need to be considered to evaluate its possible use in cancer therapies. The lectin domain of this enzyme was expressed by cloning the C-terminal portion of the DNA coding sequence and introducing it into Pichia pastoris for its recombinant production. Biophysical methods such as spectrofluorimetry and isothermal titration calorimetry were used to analyze the ability of the engineered protein to bind the T-antigen monosaccharides. The binding dissociation constant (Kd) of the protein-carbohydrate interaction was determined. The stability of the protein was also studied through its thermodynamic parameters of unfolding using differential scanning calorimetry. Crystallization screenings were set up using a broad variety of precipitants in order to produce crystals to be used to study the three-dimensional structure of the engineered protein using X-ray diffraction. The crystals that were grown were taken to the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to carry out the diffraction experiments. Although we were able to collect data up to a resolution of 2.8 Å (854,648 reflections) all the crystals we have examined so far were found to be twinned making the assignment of a definitive space group uncertain. We are currently working on correcting this problem using both the appropriate software and attempting to grow better crystals. Our goal is to produce an engineered human protein that specifically recognizes cancer specific carbohydrates and is thus suitable for protein therapeutics applied in drug-delivery methods for cancer treatment. The present structural and biophysical data are the prerequisite for future studies regarding the biological and clinical properties of the lectin. [1] Stowell, S. R. Tongzhong J. and Cummings R. D. Protein Glycosylation in Cancer. Annu Rev Pathol 2015. 10: 473–510. [2] Sharon, N., and Lis, H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004. 14: 53–62. [3] Berois, N., Mazal, D. et al. UDP-N-Acetyl-D-Galactosamine: N-acetylgalactosaminyltransferase-6 as a New Immunohistochemical Breast Cancer Marker. Journal of Histochemistry & Cytochemistry. 2006. 54(3): 317–328
Pegylated silica nanoparticles: cytotoxicity and macrophage uptake
No full textHere, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40Â nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to the silica surface, the reproducibility of the synthesis and the narrow size-dispersion. In view of clarifying the SNP interaction with biological environments, we first assessed the SNP reactivity after the incubation with two cell lines (macrophages RAW 264.7 and primary human fibroblasts), observing a reduced toxicity of pegylated SNPs compared to the bare ones. Then, we investigated the effect of the protein adsorption on the SNP surface using the model serum protein, bovine serum albumin (BSA). We found that the protein adsorption takes place more heavily on poorly pegylated SNPs, promoting the uptake of the latter by macrophages and leading to an increased mortality of these cells. To better understand this mechanism by means of flow cytometry, the dye Ru(bpy)3Cl2 was incorporated in the SNPs. The overall results highlight the SNP potentialities as a drug delivery system, thanks to the low interactions with the macrophages
Structural studies of a Tremella fuciformis mushroom lectin: a novel protein with antitumoral properties
No full textLectins are carbohydrate-binding proteins of non-immune origin, widely distributed in all the living organisms. They play a role in different biological processes and, due to their carbohydrate specific binding, some lectins have been found to possess antitumoral properties. A particular case is the recognition of the T-antigenic determinant (Galβ1-3GalNAcα) on the malignant cell surfaces, which is present in 90% of human carcinomas. Upon binding to the carbohydrate, some lectins can cause apoptosis, cytotoxicity, inhibition of tumour growth, thus preventing the proliferation of tumoral cells. Considering the fact that these carbohydrates are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells. Although the function and the biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and in the light of this, the crude extract of this fungus was examined to assess the presence of lectins. A lectin of 11 KDa, named TFL, was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. The best crystals were grown in 0.1 M TRIS pH 8.5, 1.5 potassium phosphate dibasic, 1 % DMSO and the final data sets were collected at the ESRF of Grenoble, revealing the three-dimensional structure of the protein. TFL belongs to the space group P1211 and the cell parameters of the crystal are the following: a = 61.62 Å, b = 61.83 Å, c = 67.84 Å, with β = 106.87 °. The protein is a monomer, composed of six β-sheets (from A to F) that arrange to form the so called “β-barrel”. In addition, two α-helixes, named H1 and H2 can be recognized in the structure, which is also stabilized by the presence of two disulphide bridges that connect Cys 54 to Cys 70 and Cys 97 to Cys 100, respectively. Thermal protein stability was analyzed by means of differential scanning calorimetry, revealing that TFL does not undergo a two-state unfolding process and its denaturation is reversible, a feature that has been rarely observed for lectins before. In addition, chemical and pH-induced unfolding were investigated using fluorescence spectroscopy, highlighting the high stability of TFL in a wide range of conditions. Exploiting isothermal titration calorimetry, more information about sugar-binding were acquired, identifying N-acetylgalactosamine as the best candidate, which has micromolar affinity for TFL. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and interestingly, it seems to reduce skin malignant cells migration and to have a positive effect in the upregulation of certain genes involved in cancer arresting (SBPP1). In addition, immunomodulatory activity of TFL was analyzed by means of ELISA essay, resulting in the up-regulation of the most important inflammatory markers (IL-6, TNFα), that could be usefully exploited in order to kill intracellular microorganisms and to help the human body to develop a stronger tumoral resistance. In conclusion, this work provides a new and interesting insight into cancer treatment, being an essential prerequisite for future in vitro and in vivo experiments. The encouraging results obtained so far, alongside with the fact that TFL has been produced also heterologously in E.coli, pave the way for further studies, including the feasibility to perform mutagenesis and to explore the possibility to further encapsulate the protein inside tailor-made nanoparticles, with the aim of reducing the amount of protein used, increasing its therapeutical efficiency, and preventing side effects
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
TREATMENT OF SKELETAL DISEASES WITH A NATURALLY DERIVED ANTIOXIDANT AND BISPHOSPHONATE EMBEDDED NANOPARTICLES
No full textSkeletal disorders are degenerative diseases causing progressive disability and are becoming more and more prevalent in our society. Bone is a special tissue able to support load and stress and is continuously renewed thanks to bone turnover mediated by the well coordinated activity of osteoblasts and osteoclasts. Many metabolic bone diseases are characterized by an imbalance of the activity of bone cells and bone turnover that could be estimated through the evaluation of laboratory data analysis. In addition to metabolic disorders, bone tissue can be impairs by genetic diseases such as osteogenesis imperfect. In addition, Osteoarthritis (OA), the most prevalent musculoskeletal pathology, is predominantly characterized by the progressive degradation of articular cartilage due to an imbalance between anabolic and catabolic processes. Age-related changes that occur in articular are thought to represent a major risk factor for OA development. Therefore, skeletal diseases have been associated to defective differentiation pathways of progenitor stem cells (PSCs) to produce osteoblasts or chondrocytes. RUNX2 as well as SOX9 are transcription factors 202 responsible of commitment of PSCs in osteoblasts and chondrocytes respectively. In order to evaluate the possibility to affect and improve osteoblasts or chondrocytes commitment we assayed a naturally derived antioxidant and a bisphosphonate (clodronate) embedded nanoparticles in vivo (murine model) and in vitro (cell line) respectively. Antioxidant molecule was embedded into PLGA (poly lactic-co-glycolic acid) with the emulsion evaporation method. Bisphosphonate nanoparticles were prepared using chitosan and hyaluronic acid applying the ionotropic gelation method
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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