Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.     

Fatty acid composition and biological activities of Isochrysis galbana T-ISO,Tetraselmis sp. and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries

Organic and water extracts of Isochrysis galbana T-ISO (=Tisochrysis lutea), Tetraselmis sp. and Scenedesmus sp. were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC). I. galbana T-ISO had the highest TPC (3.18 mg GAE g^?1) and radical scavenging activity, with an IC₅₀ value of 1.9 mg mL^?1 on the acetone extract. The extracts exhibited a higher ability to chelate Fe²⁺ than Cu²⁺, and the maximum Fe²⁺ chelating capacity was observed in the hexane extract of Scenedesmus sp. (IC₅₀=0.73 mg mL^?1) and Scenedesmus sp. (IC₅₀?=?0.73 mg mL^?1). The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC₅₀ values of 0.11 and 0.15 mg mL^?1, respectively, and in the water extract of I. galbana (IC₅₀?=?0.16 mg mL^?1). The acetone extract of I. galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC₅₀?=?81.3 μg mL^?1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL^?1). All species presented a highly unsaturated fatty acids profile. Results suggest that these microalgae, particularly I. galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae.     

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolyticaIly inactive, fluid-phase C5b-9 complexes, Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3BI, 3Dg, and 2F3), sheep (clones 3Dg and 2F3) and guinea-pig (clone 3D8) neoantigens were identified . Three of four tested clones (3D8, 2F3, IA12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.     

Functional unit influence on building life cycle assessment

Purpose

The building sector is one of the most relevant sectors in terms of environmental impact. Different functional units (FUs) can be used in life cycle assessment (LCA) studies for a variety of purposes. This paper aimed to present different FUs used in the LCA of buildings and evaluate the influence of FU choice and setting in comparative studies.

Methods

As an example, we compared the “cradle to grave” environmental performance of four typical Brazilian residential buildings with different construction typologies, i.e., multi-dwelling and single dwelling, each with high and basic standards. We chose three types of FU for comparison: a dwelling with defined lifetime and occupancy parameters, an area of 1 m² of dwelling over a year period, and the accommodation of an occupant person of the dwelling over a day.

Results and discussion

The FU choice was found to bias the results considerably. As expected, the largest global warming indicator (GWi) values per dwelling unit and occupant were identified for the high standard dwellings. However, when measured per square meter, lower standard dwellings presented the largest GWi values. This was caused by the greater concentration of people per square meter in smaller area dwellings, resulting in larger water and energy consumption per square meter. The sensitivity analysis of FU variables such as lifetime and occupancy showed the GWi contribution of the infrastructure more relevant compared with the operation in high and basic standard dwellings. The definition of lifetime and occupancy parameters is key to avoid bias and to reduce uncertainty of the results when performing a comparison of dwelling environmental performances.

Conclusions

This paper highlights the need for adequate choice and setting of FU to support intended decision-making in LCA studies of the building sector. The use of at least two FUs presented a broader picture of building performance, helping to guide effective environmental optimization efforts from different approaches and levels of analysis. Information regarding space, time, and service dimensions should be either included in the FU setting or provided in the building LCA study to allow adjustment of the results for subsequent comparison.

     

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.     

An optimization study of solid-state fermentation: xanthophylls extraction from marigold flowers

Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).