Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.     

Application of alternative fixatives to formalin in diagnostic pathology

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.     

New Insights in the Life Cycle and Epidemics of Phytophthora porri on Leek

White tip, caused by Phytophthora porri, is a devastating disease in the autumn and winter production of leek (Allium porrum) in Europe. This study investigated the disease cycle of P. porri in laboratory and field conditions. Oospores readily germinated in the presence of non‐sterile soil extract at any temperature between 4 and 22°C, with the formation of sporangia which released zoospores. The zoospores survived at least 7 weeks in water at a temperature range of 0 till 24°C. Microscopic examinations revealed that zoospores encysted and germinated on the leek leaf surface and hyphae entered the leaf directly through stomata or by penetrating via appressoria. Oospores were formed in the leaves within 6 days, while sporangia were not produced. By monitoring disease progress in fields with a different cropping history of leek, it could be deduced that P. porri survives in soil for up to 4 years. Disease progress during three consecutive years was correlated with average daily rainfall in the infection period. Disease incidence on leek was reduced when rain splash was excluded by growing the plants in an open hoop greenhouse. Based on these findings, we propose a disease cycle for P. porri in which oospores germinate in puddles, and zoospores reach the leaves by rain splash and survive in water in the leaf axils, from where they infect the plant by direct penetration or via stomata. When conditions become unfavourable, oospores are produced in the leaves which again reach the soil when leaves decay. Secondary spread of the disease by sporangia does not seem to be important.     

Assessment of microbial diversity in biofilms recovered from endotracheal tubes using culture dependent and independent approaches

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.     

TGFbeta protects mesoangioblasts from apoptosis via sphingosine kinase-1 regulation

Mesoangioblasts are vessel-derived progenitor cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. Here we examined the role of transforming growth factor-beta (TGFbeta), a pleiotropic cytokine that plays a major role in development and specifically induces smooth muscle differentiation of mesoangioblasts, in the regulation of death and survival of these cells. TGFbeta exerts a marked anti-apoptotic action in mesoangioblasts with a mechanism involving regulation of sphingosine kinase 1 (SphK1), one of the isoforms responsible for S1P formation. Treatment with the cytokine efficaciously protected mesoangioblasts from apoptosis induced by serum starvation or staurosporine treatment assessed by various means such as activation of caspase-3, determination of cytoplasmic histone-associated-DNA-fragments and PE-AnnexinV staining. The protective action of TGFbeta from staurosporine-induced apoptosis was strongly reduced when the SphK activity was inhibited by drugs, when SphK1 but not SphK2 was downregulated by specific siRNA and when a SphK1 dominant negative mutant was overexpressed. Staurosporine treatment induced down-regulation of both SphK isoforms and TGFbeta rescued SphK1 but not SphK2 expression. Interestingly, TGFbeta strongly enhanced SphK activity during staurosporine-induced cell death. Both TGFbeta-induced SphK1 up-regulation and TGFbeta anti-apoptotic action were found to be dependent on p42/44 MAPK activation.     

Characterization of fungal soil communities by F-RISA and arbuscular mycorrhizal fungi fromAraucaria angustifolia forest soils after replanting and wildfire disturbances

The Fungal Ribosomallntergenic Spacer Analysis (F-RlSA) was used to characterize soil fungal communities from three Cecosystems ofAraucaria angustifolia from Brazil: a native forest and two replanted forest ecosystems, one of them with a past history of wildfire. The arbuscular mycorrhizal fungi (AMF) infection was evaluated inAraucaria roots of 18-monthold axenic plants previously inoculated with soils collected from those areas in a greenhouse experiment. The principal componentanalysis of F-RISA profiles showed different soil fungal community betweenthe three studied areas. Sixty three percent of F-RISA fragments amplified in the soil and the substrate samples presented lengths between 500 and 700 bp. The number of Operational Taxonomic Units (OTUs) was 34 for soil and 38 for substrate, however, more fragments were detected in soil (214) than in substrate (163). Anin silico F-RISA analysis to compare our data with ITSI-5.8S-ITS2 sequences from NCBI database showed the presence of Ascomycota, Basidiomycota and Glomeromycota among the soil and substrate fungal communities. AMF infection was higher in plants inoculated with soil from the native forest and the replanted forest with wildfire, both presenting similar chemical characteristics but with different disturbance levels. These results indicate that soil chemical composition may influence the soil fungal community structures rather than the anthropogenicor fire disturbances.     

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.     

Isozyme gene markers in Vicia faba L.

Summary This study was conducted to assess the genetic basis of the variability observed for the glutamate oxaloacetate transaminase (GOT), Superoxide dismutase (SOD), esterase (EST), and malate dehydrogenase (MDH) isozyme systems in different open-pollinated Vicia faba varieties. Individual plants showing contrasting zymogram patterns were simultaneously selfed and cross-combined. Crossing was unsuccessful in producing progeny, and only selfed progenies were suitable for genetical analysis of isozyme variability. Three zones of GOT activity were made visible. The isozyme of GOT-2 and GOT-3 zones were dimeric and under the control of three alleles at the Got-2 locus and two alleles at the Got-3 locus, respectively. The isozymes of the GOT-1 zone did not show any variability. Three zones of SOD isozyme activity were made visible. The isozymes occurring in the SOD-1 (chloroplastic isozyme form) and SOD-2 (cytosol isozyme form) zones were dimeric and under the control of two alleles at the Sod-1 and Sod-2 loci. The isozyme visualized in the SOD-3 zone (mitochondrial isozyme form) were tetrameric and under the control of two alleles at the Sod-3 locus. Apparently the isozymes made visible in the most anodal esterase zones EST-1, EST-2, and EST-3 were monomeric, and the occurrence of two alleles at each of two different loci explained the variability observed in the EST-2 and EST-3 zones. For MDH, only two five-banded zymogram pattern types were found, and every selfed progeny showed only one of the two zymogram type, indicating that each individual possessed fixed alleles at the loci controlling MDH isozyme. Got-2, Got-3, Sod-1, Sod-2, and Sod-3 appear to be five new isozyme gene markers that can be useful in Vicia faba breeding for linkage study, varietal fingerprinting, outcrossing rate estimate, and indirect selection for quantitative characters.