Purification and Characterization of a New D-Galactose-Specific Lectin from the Housefly, Musca domestica, and Its Antiproliferative Effect on Human K562 and MCF-7 Tumor Cells

In the present work, a D-galactose-specific lectin with novel N-terminal sequence was purified from Musca domestica L. (Diptera: Muscidae) pupae. The purification was performed using affinity chromatography, ultra-filtration, and HPLC. The haemagglutinating activity of M. domestica lectin was specifically inhibited by D-galactose. The haemagglutinating activity of this lectin was stable at temperatures up to 65° C and in pH ranging from 4 to 8. Salts including FeCl3 and MnCl2 inhibited the haemagglutinating process, whereas NaCl, KCl, CaCl2, MgCl2, ZnCl2, and AlCl3 did not. By SDS-PAGE, purified M. domestica pupae lectin yielded a single band with a molecular weight of 40 kDa, with or without reduction of β-mercaptoethanol, and it could be stained with Alcian Blue 8 GX. The morphology of purified lectin was observed by atomic force microscopy, which indicated that M. domestica lectin was an 8.27 nm high, globular shaped glycoprotein with a 1.41 nm high polysaccharide chain. In addition, antiproliferative activity of this lectin against tumor cells K562 and MCF-7 was determined with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, which showed that the antiproliferative process was time- and dose-dependent with an IC50 of 5.7 and 6.7 at 24 h, 5.5 and 6.4 at 36 h, 5.2 and 6.5 µM at 48 h, respectively

Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages

The activation of P2X7 is a well-known stimulus for the NLRP3-caspase 1 inflammasome and subsequent rapid IL-1β secretion from monocytes and macrophages. Here we show that positive allosteric modulators of P2X7, ginsenosides, can enhance the release of three important cytokines, IL-1β, IL-6 and TNF-α from LPS-primed rodent macrophages using the J774 mouse macrophage cell line and primary rat peritoneal macrophages. We compared the immediate P2X7 responses in un-primed and LPS-primed macrophages and found no difference in calcium response amplitude or kinetics. These results suggest that under inflammatory conditions positive allosteric modulators are capable of increasing cytokine secretion at lower concentrations of ATP, thus boosting the initial pro-inflammatory signal. This may be important in the control of intracellular infections

Ginsenosides act as positive modulators of P2X4 receptors

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/ Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP 2 or ATP 1 ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Photocalorimetry: Development of methods to assess the photostability of pharmaceutical compounds

The main focus of the research presented in this thesis is to develop a robust and easy-to-use photocalorimeter to allow a quantitative assessment of the photostability of pharmaceutical compounds. As a result, a novel photocalorimeter was successfully developed. Initially a Xe-arc lamp was used as a light source, but this was found to be problematic, rendering quantitative analysis of data challenging. A novel approach using light-emitting diodes (LEDs) as a light source was developed; this approach demonstrated much potential in photostability testing. Using the prototype systems a suitable method to allow measurement of the radiant energy delivered to the sample during a photochemical process was investigated. The best system appeared to be the photodegradation of 2-nitrobenzaldehyde (2NB). Although promising as a test and reference reaction, it was found that 2NB was sensitive to non-photochemical processes and the reaction was complex which means further work on its application in this area must be undertaken. The application of chemometric analysis as an approach to interpret complexity in Isothermal Calorimetric data was then studied. A three-step consecutive reaction was used to demonstrate the applicability of principal component analysis to determine the number of reaction steps and reaction parameters in a process. The application of photocalorimetry for the study of a known photosensitive drug, nifedipine was investigated. The photolysis of nifedipine in solution was studied under full spectrum lighting and under specific wavelengths for the determination of causative wavelength (s). The data showed the photodegradation of nifedipine to be particularly sensitive at 360 nm. No significant photosensitivity was detected above 370 nm. Finally a novel autobalance power supply, currently under development, designed to improve the performance of the photocalorimeter is described

Cytoprotective effect of methanolic extract of Nardostachys jatamansi against hydrogen peroxide induced oxidative damage in C6 glioma cells

Oxidative stress has been implicated as an important factor in the process of neurodegeneration and hydrogen peroxide (H2O2) is one of the most important precursors of reactive oxygen species (ROS), responsible for many neurodegenerative diseases. This study used extracts from Nardostachys jatamansi rhizomes, known for nerve relaxing properties in Ayurvedic medicine, to ascertain their protective role in H2O2-induced oxidative stress in C6 glioma cells. The protective effect of methanolic, ethanolic and water extracts of N. jatamansi (NJ-MEx, NJ-EEx and NJ-WEx respectively) was determined by MTT assay. NJ-MEx significantly protected against H2O2 cytotoxicity when cells were pretreated for 24 h. The level of antioxidant enzymes, catalase, superoxide dismutase (Cu-ZnSOD), glutathione peroxidase (GPx), and a direct scavenger of free radicals, glutathione (GSH), significantly increased following pre-treatment with NJ-MEx. Lipid peroxidation (LPx) significantly decreased in NJ-MEx-pretreated cultures. The expression of a C6 differentiation marker, GFAP (glial fibrillary acidic protein), stress markers HSP70 (heat shock protein) and mortalin (also called glucose regulated protein 75, Grp75) significantly decreased when cells were pre-treated with NJ-MEx before being subjected to H2O2 treatment as shown by immunofluorescence, western blotting and RT-PCR results. The present study suggests that NJ-MEx could serve as a potential treatment and/or preventive measure against neurodegenerative diseases

Isolation, purification and characterization of an N-acetyl-D-lactosamine binding mitogenic and anti-proliferative lectin from tubers of a cobra lily Arisaema utile Schott

Lectins are the carbohydrate-binding proteins of non-immune origin which have been the subject of intense investigation over the last few decades owing to the variety of interesting biological properties. Most of the lectins which have been purified and characterized from plants have been obtained from dicotyledons. In the present study a lectin was purified from tubers of a monocot plant Arisaema utile (AUL) Schott by affinity chromatography on asialofetuin- linked amino activated silica beads. AUL gave a single band in SDS-PAGE at pH 8.3 corresponding to subunit Mr 13.5 kDa. The native molecular mass of AUL was 54 kDa suggesting a homotetrameric structure. AUL gave multiple bands in isoelectric focusing and in native PAGE at pH 8.3. AUL was inhibited by N-acetyl-D-lactosamine (Lac NAc), a disaccharide and asialofetuin, a complex desialylated serum glycoprotein. When treated with denaturing agents, the lectin was stable in the presence of urea (3 M), thiourea (4 M) and guanidine HCl (4 M). AUL was a glycoprotein with a carbohydrate content of 1.2%. Complete loss of activity was observed upon modification of tryptophan residues of the lectin. The activity was reduced to 25% after modification of tyrosine. Chemical modification of arginine, histidine, serine and cysteine residues of AUL did not affect its activity. Using Far UV CD spectra the estimated secondary structure was 37% α-helix, 25% β-sheet and 38% random contributions. The lectin showed potent mitogenic response towards human lymphocytes. In vitro anti-proliferative assay using 11 human cancer cell lines resulted in 50% inhibition of six cell lines viz. SW-620, HCT-15, SK-N-SH, IMR-32, Colo-205 and HT-29 at 38, 42, 43, 49, 50 and 89 μg/ml, respectively

Role of glass structure in defining the chemical dissolution behavior, bioactivity and antioxidant properties of zinc and strontium co-doped alkali-free phosphosilicate glasses

We investigated the structure-property relationships in a series of alkali-free phosphosilicate glass compositions co-doped with Zn2+ and Sr2+. The emphasis was laid on understanding the structural role of Se2+ and Zn2+ co-doping on the chemical dissolution behavior of glasses and its impact on their in vitro bioactivity. The structure of glasses was studied using molecular dynamics simulations in combination with solid state nuclear magnetic resonance spectroscopy. The relevant structural properties are then linked to the observed degradation behavior, in vitro bioactivity, osteoblast proliferation and oxidative stress levels. The apatite-forming ability of glasses has been investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy-energy-dispersive spectroscopy after immersion of glass powders/bulk in simulated body fluid (SBF) for time durations varying between 1 h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the glasses exhibit hydroxyapatite formation on their surface within 1-3 h of their immersion in SBF. The cellular responses were observed in vitro on bulk glass samples using human osteosarcoma MG63 cell line. The dose-dependent cytoprotective effect of glasses with respect to the concentration of zinc and strontium released from the glasses is also discussed. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved