Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Platelet GPIb complex as a target for anti-thrombotic drug development

Specific inhibition of platelet function is a major target of anti-thrombotic drug research. Platelet receptors are both accessible and specific but have multiple functions often linked to a wide range of ligands. GPIb complex is best known as a major platelet receptor for von Willebrand factor essential for platelet adhesion under high shear conditions found in arteries and in thrombosis. Recent animal studies have supported inhibition of GPIb as a good candidate for anti-thrombotic drug development with several classes of proteins showing important specific effects and the required discrimination between roles in haemostasis and thrombosis is important to protect against bleeding complications. These include antibodies, several classes of snake venom proteins, mutant thrombin molecules and peptides affecting subunit interactions. However, due to the nature of its receptor-ligand interactions involving large protein-protein interfaces, the possibility of developing classic pharmaceutical inhibitors for long term (and perhaps oral) treatment is still unclear, and additional information about structural interactions and signalling mechanisms is essential.

Strengthening of laterality of verbal and visuospatial functions during childhood and adolescence

Cognitive functions in the child's brain develop in the context of complex adaptive processes, determined by genetic and environmental factors. Little is known about the cerebral representation of cognitive functions during development. In particular, knowledge about the development of right hemispheric (RH) functions is scarce. Considering the dynamics of brain development, localization and lateralization of cognitive functions must be expected to change with age. Twenty healthy subjects (8.6-20.5 years) were examined with fMRI and neuropsychological tests. All participants completed two fMRI tasks known to activate left hemispheric (LH) regions (language tasks) and two tasks known to involve predominantly RH areas (visual search tasks). A laterality index (LI) was computed to determine the asymmetry of activation. Group analysis revealed unilateral activation of the LH language circuitry during language tasks while visual search tasks induced a more widespread RH activation pattern in frontal, superior temporal, and occipital areas. Laterality of language increased between the ages of 8-20 in frontal (r = 0.392, P = 0.049) and temporal (r = 0.387, P = 0.051) areas. The asymmetry of visual search functions increased in frontal (r = -0.525, P = 0.009) and parietal (r = -0.439, P = 0.027) regions. A positive correlation was found between Verbal-IQ and the LI during a language task (r = 0.585, P = 0.028), while visuospatial skills correlated with LIs of visual search (r = -0.621, P = 0.018). To summarize, cognitive development is accompanied by changes in the functional representation of neuronal circuitries, with a strengthening of lateralization not only for LH but also for RH functions. Our data show that age and performance, independently, account for the increases of laterality with age.

Mammalian pre-mRNA 3' end processing factor CF I m 68 functions in mRNA export

Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3' end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3' end processing complex, CF I(m)68, stimulates mRNA export. CF I(m)68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF I(m)68 may act as a novel adaptor for NXF1/TAP, we show that CF I(m)68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF I(m)68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3' end processing factor CF I(m)68 in mRNA export.

Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions

The evolutionarily conserved Mre11/Rad50/Nbs1 (MRN) complex is involved in various aspects of meiosis. Whereas available evidence suggests that the Mre11 nuclease activity might be responsible for Spo11 removal in Saccharomyces cerevisiae, this has not been confirmed experimentally. This study demonstrates for the first time that Mre11 (Schizosaccharomyces pombe Rad32(Mre11)) nuclease activity is required for the removal of Rec12(Spo11). Furthermore, we show that the CtIP homologue Ctp1 is required for Rec12(Spo11) removal, confirming functional conservation between Ctp1(CtIP) and the more distantly related Sae2 protein from Saccharomyces cerevisiae. Finally, we show that the MRN complex is required for meiotic recombination, chromatin remodeling at the ade6-M26 recombination hot spot, and formation of linear elements (which are the equivalent of the synaptonemal complex found in other eukaryotes) but that all of these functions are proficient in a rad50S mutant, which is deficient for Rec12(Spo11) removal. These observations suggest that the conserved role of the MRN complex in these meiotic functions is independent of Rec12(Spo11) removal.

Knudsen effusion mass spectrometric determination of the complex vapor composition of samarium, europium, and ytterbium bromides

RATIONALE The vaporization of Sm, Eu, and Yb tri- and dibromides is accompanied by decomposition and disproportionation reactions. These result in complex vapor compositions whose analysis is an intricate problem for experimentalists. Approaches have been developed to interpret mass spectra and accurately determine the vapor composition of thermally unstable compounds. METHODS A sector type magnet instrument was used. A combined ion source allowed the study of both the molecular and ionic vapor compositions in the electron ionization (EI) and the thermionic emission (TE) modes. The methodological approaches were based on a joint analysis of the ionization efficiency functions, the temperature and time dependences of the ion currents, and special mathematical data evaluation. RESULTS The vaporization of SmBr3, YbBr3, SmBr2, EuBr2, and YbBr2 was studied in the temperature range of 850–1300 K. An initial stage of incongruent vaporization was observed in the case of the tribromides, SmBr2, and YbBr2. This eventually changed to a congruent vaporization stage. Various neutral (Ln, Br, Br2, LnBr, LnBr2, LnBr3, Ln2Br4, Ln2Br5, and Ln2Br6) and charged (Br–, LnBr3–, LnBr4–) species were detected at different vaporization stages. CONCLUSIONS The quantitative vapor composition of Sm, Eu, and Yb tri- and dibromides was determined. It was found that only EuBr2 was stable in the studied temperature range. The developed approaches can be useful in the case of other thermally unstable compounds.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo , while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Porcine cathelicidins efficiently complex and deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α responses.

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine-rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide-DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid-complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Higher Visual Functions in the Upper and Lower Visual Fields: a pilot study in healthy subjects

Visual perception is not identical in the upper and lower visual hemifields. The mechanisms behind this difference can be found at the retinal, cortical, or higher attentional level. In this study, a new visual test battery, that involves real-time comparisons of complex visual stimuli, such as shape of objects, and speed of moving dot patterns, in the upper and lower visual hemifields, is presented. This study represents, to our knowledge, the first to implement such a visual test battery in an immersive environment composed of a hemisphere, in order to present visual stimuli in precise regions of the visual field. Ten healthy volunteers were tested in this pilot study. The results showed a higher accuracy in the image matching when the visual test was performed in the lower visual hemifield.