High lipid content of irradiated human melanoma cells does not affect cytokine-matured dendritic cell function

Gamma irradiation is one of the methods used to sterilize melanoma cells prior to coculturing them with monocyte-derived immature dendritic cells in order to develop antitumor vaccines. However, the changes taking place in tumor cells after irradiation and their interaction with dendritic cells have been scarcely analyzed. We demonstrate here for the first time that after irradiation a fraction of tumor cells present large lipid bodies, which mainly contain triglycerides that are several-fold increased as compared to viable cells as determined by staining with Oil Red O and BODIPY 493/503 and by biochemical analysis. Phosphatidyl-choline, phosphatidyl-ethanolamine and sphingomyelin are also increased in the lipid bodies of irradiated cells. Lipid bodies do not contain the melanoma-associated antigen MART-1. After coculturing immature dendritic cells with irradiated melanoma cells, tumor cells tend to form clumps to which dendritic cells adhere. Under such conditions, dendritic cells are unable to act as stimulating cells in a mixed leukocyte reaction. However, when a maturation cocktail composed of TNF-alpha, IL-6, IL-1beta and prostaglandin E2 is added to the coculture, the tumor cells clumps disaggregate, dendritic cells remain free in suspension and their ability to efficiently stimulate allogeneic lymphocytes is restored. These results help to understand the events following melanoma cell irradiation, shed light about interactions between irradiated cells and dendritic cells, and may help to develop optimized dendritic cell vaccines for cancer therapy.     

Rio Tinto as a niche for acidophilus enzymes of industrial relevance

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.     

Purification and properties of agmatine amidinohydrolase of Evernia prunastri

An agmatine amidinohydrolase (EC 3.5.3.11) has been purified from Evernia prunastri (L.) Ach. thallus incubated on 40 m M L-arginine at 26°C in the dark. The enzyme was purified 485-fold with an overall yield of 55%. It shows a pH optimum of 6.9, a temperature optimum at 35–40°C and a molecular mass (weight) of about 320 000. The Evernia hydrolase is significantly activated by L-arginine, L-ornithine and putrescine for agmatine concentrations lower than 14 m M and inhibited for agmatine concentrations producing inhibition by an excess of substrate. Urea was always a powerful inhibitor of the enzyme. The K_m for agmatine was estimated to be 6.4 m M .     

A multianalyte flow electrochemical cell: application to the simultaneous determination of carbohydrates based on bioelectrocatalytic detection

A multianalyte flow electrochemical cell (MAFEC) for bioanalysis is constructed, characterised and used for simultaneous carbohydrate analysis incorporating mediated amperometric enzyme electrodes. Although multidetection schemes can be addressed with microfabricated systems, it is demonstrated that a "meso" analytical device of low cost can give answers to traditional simultaneous multianalysis problems, being robust, and easy to construct and operate. The cell operates as a radial flow thin-layer device and can achieve mass transport controlled response for fast electrochemical reactions. When appropriate enzymatic electrodes are used the response becomes kinetically limited, but still shows a better than 5% R.S.D. for response to different sugars analysed. All the enzymatic sensors are mediated with different osmium compounds appropriate for each enzyme's mechanism (NAD or PQQ dehydrogenases) in some cases combining multienzyme sensors. All sensors were optimised so that different sugars do not produce interferences to other sensors. Matrix interferences were kept low by operating all sensors at or below 150 mV versus Ag/AgCl. The integrated system was used for the simultaneous detection of fructose, sucrose, glucose, galactose, and lactose, fully characterising the system for these analytes (sensitivity, dynamic range). Cross referenced calibration curves were used for signal treatment and interpretation and it was possible to analyse real juice and milk samples with results agreeing with the standard enzymatic methods for the same analyses with a sampling frequency of more than 100 h(-1).     

Transcriptional phase variation at the flhB gene of Pseudomonas putida DOT-T1E is involved in response to environmental changes and suggests the participation of the flagellar export system in solvent tolerance

Frameshift mutations in a poly(G) track at the flhB gene of Pseudomonas putida DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of P. putida KT2440, which does not exhibit a poly(G) track at the flhB gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in P. putida DOT-T1E, so that growth conditions favoring a functional flhB gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.     

Cycloaddition reactions of pristine and endohedral fullerene molecules: possible anticancer activity

Epoxide of oestradiol is one of the main risk factors for the genesis and evolution of breast cancer; hence, in recent years there has been considerable interest in the investigation of new inhibitors capable of reducing its carcinogenic activity. The aim of this article is to study the [2 + 2] cycloaddition reaction of epoxide of oestradiol in different pristine (C₇₆ and D_5h-C₈₀) and endohedral metallofullerene (C₇₂@Sc₂C₂, C₇₆@Sc₂ and C₈₀@Sc₂) by means of molecular electrostatic potential (MEP) topological analysis. Different from other molecular scalar fields, MEP topology enables to find minima related to lone pairs and π electrons, therefore, this molecular scalar field is appropriate to identify the most reactive sites. In consonance with our results, it was found that C₈₀ was the best candidate to carry out the epoxide of oestradiol cycloaddition since more stable adducts were obtained. Furthermore, it is expected that more than one oestradiol epoxide molecule will be added to C₈₀, forasmuch as C₈₀ reactivity is enhanced once the adduct is formed. The study was carried through DFT framework included in the Gaussian 09 package (MPWB95/6-31G(d,p)).     

Differential bacterial growth kinetic and nitrification of fisheries wastewaters containing high ammonium and organic matter concentration by using pure oxygen

Nitrification with pure O of fisheries wastewaters high in N-NH4+ (0.2-1g/l) and in organic matter (1-2gCOD/l) gave specific growth rates for the bacteria degrading organic matter of 0.24h and for nitrifying bacteria of 0.15h. Average organic matter abatement was 75% whereas N-NH (0.7g/l as initial concentration) oxidation to nitrite and nitrate was 20%.     

A Trichoderma atroviride stress‐activated MAPK pathway integrates stress and light signals

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.     

Rapid postglacial diversification and long‐term stasis within the songbird genus Junco: phylogeographic and phylogenomic evidence

Natural systems composed of closely related taxa that vary in the degree of phenotypic divergence and geographic isolation provide an opportunity to investigate the rate of phenotypic diversification and the relative roles of selection and drift in driving lineage formation. The genus Junco (Aves: Emberizidae) of North America includes parapatric northern forms that are markedly divergent in plumage pattern and colour, in contrast to geographically isolated southern populations in remote areas that show moderate phenotypic divergence. Here, we quantify patterns of phenotypic divergence in morphology and plumage colour and use mitochondrial DNA genes, a nuclear intron, and genomewide SNPs to reconstruct the demographic and evolutionary history of the genus to infer relative rates of evolutionary divergence among lineages. We found that geographically isolated populations have evolved independently for hundreds of thousands of years despite little differentiation in phenotype, in sharp contrast to phenotypically diverse northern forms, which have diversified within the last few thousand years as a result of the rapid postglacial recolonization of North America. SNP data resolved young northern lineages into reciprocally monophyletic lineages, indicating low rates of gene flow even among closely related parapatric forms, and suggesting a role for strong genetic drift or multifarious selection acting on multiple loci in driving lineage divergence. Juncos represent a compelling example of speciation in action, where the combined effects of historical and selective factors have produced one of the fastest cases of speciation known in vertebrates.