Differential expression of MHC class II molecules in highly metastatic breast cancer cells is mediated by the regulation of the CIITA transcription Implication of CIITA in tumor and metastasis development

We analyzed the differential gene expression between variants of MDA-MB-435 human breast cancer cell line that share an identical genetic background but have different metastatic ability. The major histocompatibility complex class II was found down-regulated in highly metastatic cells and correlated with MHC transactivator (CIITA) expression. Constitutive CIITA expression observed in poorly metastatic is driven by promoters III and IV of CIITA gene. Conversely, both promoters were ineffective in highly metastatic cells. The MHC class II and CIITA expression was restored in these cells upon stimulation with IFNgamma or by the treatment with a hypomethylating agent. Both treatments induced USF-1 and IRF binding complexes to promoter IV but only IFNgamma induced the binding of 435-Lung2 nuclear proteins to an ARE-1 site at the promoter III. Neither Southern blot nor bisulfite sequencing of promoter IV demonstrated strong hypermethylation of this promoter at the IFNgamma-responsive elements such as GAS, E-box or IRF-1. We suggest that partial or hemimethylation of promoter IV is sufficient to silence the CIITA expression in highly metastatic cells and that this epigenetic mechanism is responsible for the lack of MHC-II expression. Forced CIITA expression restored the MHC-II antigen expression in 435-Lung2 cells and abrogates spontaneous lung metastasis in both SCID and nude mice but also affected the tumorigenicity in nude mice. The increase in NK cell infiltration in nude mice bearing CIITA-tumors correlated with sign of tumor cell apoptosis and the increase in the number of NK cells in the spleens, suggesting that NK cells might be responsible for the observed antitumor activity.     

COPASI--a COmplex PAthway SImulator

MOTIVATION: Simulation and modeling is becoming a standard approach to understand complex biochemical processes. Therefore, there is a big need for software tools that allow access to diverse simulation and modeling methods as well as support for the usage of these methods. RESULTS: Here, we present COPASI, a platform-independent and user-friendly biochemical simulator that offers several unique features. We discuss numerical issues with these features; in particular, the criteria to switch between stochastic and deterministic simulation methods, hybrid deterministic-stochastic methods, and the importance of random number generator numerical resolution in stochastic simulation. AVAILABILITY: The complete software is available in binary (executable) for MS Windows, OS X, Linux (Intel) and Sun Solaris (SPARC), as well as the full source code under an open source license from http://www.copasi.org.     

Probing the interaction between vesicular stomatitis virus and phosphatidylserine

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV–PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide–membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy–Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val¹⁴⁵ and His¹⁴⁸. Fabiana A.Carneiro and Pedro A. Lapido-Loureiro contributed equally to this work An erratum to this article can be found at     

Metabolic homeostasis in the human erythrocyte: in silico analysis

A detailed computer model of human erythrocyte metabolism was shown to predict three steady states, two stable and one unstable. The most extreme steady state is characterized by almost zero concentrations of all the phosphorylated intermediates. The "normal" steady state is remarkably robust in the face of large changes in the activity of most of the enzymes of glycolysis and the pentose phosphate pathway: this steady state can be viewed as an attractor towards which the system returns following a metabolic perturbation. Focus is given to three responses of the system: (1) the 'energy charge' that pertains to the concentration of ATP relative to all purine nucleotides; (2) redox power expressed as the ratio of reduced-to-total glutathione and (3) the concentration of 2,3-bisphosphoglycerate, that directly affects the oxygen affinity of haemoglobin thus affecting the main physiological function of the cell. The collapse of the normal steady state in what can be viewed topologically as a catastrophe is posited as one key element of erythrocyte senescence and it is particularly important for erythrocyte destruction in patients with an inborn enzyme deficiency.     

Environmental factors influence on chemical polymorphism of the essential oils of Lychnophora ericoides

Lychnophora ericoides is a Brazilian medicinal plant used in folk medicine as an anti-inflammatory and analgesic agent. The essential oils from leaves of two populations with and without scent, collected at 2-month intervals during an 1-year period, were analysed by GC-MS. The results were submitted to principal component and cluster analysis which allowed two groups of essential oils to be distinguished with respect to sampling site and scent: cluster I (Vianópolis site, with specimens exhibiting an aromatic scent) containing a high percentage of alpha-bisabolol (44.7-76.4%) and alpha-cadinol (10.9-23.5%), and cluster II (Cristalina site, with specimens without scent) characterised by a high content of (E)-nerolidol (31.3-47.1%) and ar-dihydro-turmerone (4.8-15.4%). The canonical discriminant analysis showed that using the data set of the seven sampling months and (E)-nerolidol and alpha-bisabolol as predictable variables, it was possible to distinguish between the samples harvested according to Cerrado seasons, dry winter (May-September) and humid summer (November-March). In addition, canonical correlation analysis between the soil sampling sites and the populations revealed a significant relationship between oil components and edaphic factors. Oxygenated sesquiterpenes and potential acidity, Al saturation, cationic exchange capacity, silt, and sand load as the first canonical variate were fairly strongly related to samples collected in Vianópolis site. On the other hand, monoterpenes and sesquiterpene hydrocarbons were strongly related to chemical balance in soils (organic matter, P and base saturation), which is related to samples at the Cristalina site. The chemovariation observed appears to be environmentally determined.     

Annual changes in the concentration of minerals and organic compounds of Quercus suber leaves

Mature leaves are the primary source of sugars, which give rise to many secondary metabolites required for plant survival under adverse conditions. In order to study the interaction of field‐grown cork oak (Quercus suber L.) with the environment, we investigated the seasonal variation of minerals and organic metabolites in the leaves, using inductively coupled plasma atomic emission spectrometry, elemental analysis and nuclear magnetic resonance spectrometry. Statistical analysis showed that the data strongly correlated with seasonal climate and were divided in three groups corresponding to: (1) spring‐early summer, (2) summer and (3) autumn‐winter. The concentration of N, P, K and leaf ash content were highest in spring (recently formed leaves), reached the minimum during the hot and dry summer and increased slightly during the rainy period of autumn‐winter. Conversely, Na, Mg and Ca concentrations were lowest in spring‐early summer and increased during summer and autumn‐winter, the Ca concentration increasing five‐fold. Two cyclitol derivatives, quinic acid and quercitol were the major organic metabolites of the leaves. Their concentration along the season followed opposite trends. While quinic acid predominated during spring‐early summer, when it contributed 12% to the leaf osmotic potential, quercitol was predominant during autumn‐winter, when its contribution to leaf osmotic potential was about 10%. This different preponderance of the two compounds is expressed by the quercitol/quinic acid ratio, which can be as low as 0.2 in early summer and as high as 9 in winter. Sucrose and glucose concentrations also increased during autumn‐winter. Evidence for the quercitol protective role in plants during stress is discussed, and on the basis of structural similarity, it is suggested that quinic acid could have an identical importance, with a protective role against heat and high irradiance. It is concluded that the marked changes in Q. suber leaf composition throughout the year could have important implications in the plant capacity to endure climatic stress.     

Similarities and differences in the thioredoxin superfamily

There is growing interest in the proteins involved in protein folding. This is mainly due to the large number of human diseases related to defects in folding, which include cystic fibrosis, Alzheimer's and cancer. However, equally important as the oxidation and concomitant formation of disulfide bridges of the extracellular or secretory proteins is the reduction and maintenance in the reduced state of the proteins within the cell. Interestingly, the proteins that are responsible for maintenance of the reduced state belong to the same superfamily as those responsible for the formation of disulfide bridges: all are members of the thioredoxin superfamily. In this article, we highlight the main features of those thioredoxin-like proteins directly involved in the redox reactions. We describe their biological functions, cytoplasmic location, mechanisms of action, structures and active site features, and discuss the principal hypotheses concerning origins of the different reduction potentials and unusual pK_a's of the catalytic residues.     

A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy

We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis.     

Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.