The stable assembly of newly synthesized PsaE into the photosystem I complex occurring via the exchange mechanism is facilitated by electrostatic interactions

Photosystem I (PSI) is a photochemically active membrane protein complex that functions at the reducing site of the photosynthetic electron-transfer chain as plastocyanin-ferredoxin oxidoreductase. PsaE, a peripheral subunit of the PSI complex, plays an important role in the function of PSI. PsaE is involved in the docking of ferredoxin/flavodoxin to the PSI complex and also participates in the cyclic electron transfer around PSI. The molecular characterization of the assembly of newly synthesized PsaE in the thylakoid membranes or in isolated PSI complexes is the subject of the present study. For this purpose the Mastigocladus laminosus psaE gene was cloned and overexpressed in Escherichia coli, and the resulting PsaE protein was purified to homogeneity by affinity chromatography. The purified PsaE was then introduced into thylakoids isolated from M. laminosus, and the newly introduced PsaE subunit saturates the membrane. The solubilization and separation of the different thylakoid protein complexes indicated that PsaE accumulates specifically in its functional location, the PSI complex. A similar stable assembly was detected when PsaE was introduced into purified PSI complexes, i.e., in the absence of other thylakoid components. This strongly indicates that the information for the stable assembly of PsaE into PSI lies within the polypeptide itself and within other subunits of the PSI complex that interact with it. To determine the nature of these interactions, the assembly reaction was performed in conditions affecting the ionic/osmotic strength. We found that altering the ionic strength significantly affects the capability of PsaE to assemble into isolated thylakoids or PSI complexes, strongly supporting the fact that electrostatic interactions are formed between PsaE and other PSI subunits. Moreover, the data suggest that the formation of electrostatic interactions occurs concomitantly with an exchange step in which newly introduced PsaE replaces the subunit present in situ.     

Evidence for the complexity of microRNA-mediated regulation in ovarian cancer: a systems approach

MicroRNAs (miRNAs) are short (～22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ～11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized.     

Isolation and characterization of stem-like cells from a human ovarian cancer cell line

Increasing evidence supports the existence of a subpopulation of cancer cells capable of self-renewal and differentiation into diverse cell lineages. These cancer stem-like or cancer-initiating cells (CICs) also demonstrate resistance to chemo- and radiotherapy and may function as a primary source of cancer recurrence. We report here on the isolation and in vitro propagation of multicellular ovarian cancer spheroids from a well-established ovarian cancer cell line (OVCAR-3). The spheroid-derived cells (SDCs) display self-renewal potential, the ability to produce differentiated progeny, and increased expression of genes previously associated with CICs. SDCs also demonstrate higher invasiveness, migration potential, and enhanced resistance to standard anticancer agents relative to parental OVCAR-3 cells. Furthermore, SDCs display up-regulation of genes associated with epithelial-to-mesenchymal transition (EMT), anticancer drug resistance and/or decreased susceptibility to apoptosis, as well as, down-regulation of genes typically associated with the epithelial cell phenotype and pro-apoptotic genes. Pathway and biological process enrichment analyses indicate significant differences between the SDCs and precursor OVCAR-3 cells in TGF-beta-dependent induction of EMT, regulation of lipid metabolism, NOTCH and Hedgehog signaling. Collectively, our results indicate that these SDCs will be a useful model for the study of ovarian CICs and for the development of novel CIC-targeted therapies.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Utilization of PVX-Cre expression vector in potato

Trait genes are usually introduced into the plant genome together with a marker gene. The last one becomes unnecessary after transgene selection and characterization. One of the strategies to produce transgenic plants free from the selectable marker is based on site-specific recombination. The present study employed the transient Cre-lox system to remove the nptII marker gene from potato. Transient marker gene excision involves introduction of Cre protein in lox-target plants by PVX virus vector followed by plant regeneration. Using optimized experimental conditions, such as particle bombardment infection method and application of P19 silencing suppressor protein, 20-27% of regenerated plants were identified by PCR analysis as marker-free. Based on our comparison of the recombination frequencies observed in this study to the efficiency of other methods to avoid or eliminate marker genes in potato, we suggest that PVX-Cre mediated site-specific excisional recombination is a useful tool to generate potato plants without superfluous transgenic sequences.     

Learning-induced modulation of oscillatory activities in the mammalian olfactory system: The role of the centrifugal fibres

In the mammalian olfactory system, oscillations related to odour representation have been described in field potential activities. Previous results showed that in olfactory bulb (OB) of awake rats engaged in an olfactory learning, odour presentation produced a decrease of oscillations in gamma frequency range (60-90 Hz) associated with a power increase in beta frequency range (15-40 Hz). This response pattern was strongly amplified in trained animals. The aim of this work was twofold: whether learning also induces similar changes in OB target structures and whether such OB response depends on its centrifugal inputs. Local field potentials (LFPs) were recorded through chronically implanted electrodes in the OB, piriform and enthorhinal cortices of freely moving rats performing an olfactory discrimination. Oscillatory activities characteristics (amplitude, frequency and time-course) were extracted in beta and gamma range by a wavelet analysis. First, we found that odour induced beta oscillatory activity was present not only in the OB, but also in the other olfactory structures. In each recording site, characteristics of the beta oscillatory responses were dependent of odour, structure and learning level. Unilateral section of the olfactory peduncle was made before training, and LFPs were symmetrically recorded in the two bulbs all along the acquisition of the learning task. Data showed that deprivation of centrifugal feedback led to an increase of spontaneous gamma activity. Moreover, under this condition olfactory learning was no longer associated with the typical large beta band. As a whole, learning modulation of the beta oscillatory response in olfactory structures may reflect activity of a distributed functional network involved in odour representation.     

LTR retrotransposons and the evolution of dosage compensation in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

Dosage compensation in Drosophila is the epigenetic process by which the expression of genes located on the single X-chromosome of males is elevated to equal the expression of X-linked genes in females where there are two copies of the X-chromosome. While epigenetic mechanisms are hypothesized to have evolved originally to silence transposable elements, a connection between transposable elements and the evolution of dosage compensation has yet to be demonstrated.     

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons,respectively

A receptor–ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type‐specific post‐receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death‐triggering factor in motoneurons through LT‐βR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT‐induced axonal elongation and branching require ERK and caspase‐9 pathways. This distinct response involves a compartment‐specific activation of LIGHT signals, with somatic activation‐inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.