Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Temporal and spatial variability in stable isotope ratios of SPM link to local hydrography and longer term SPM averages suggest heavy dependence of mussels on nearshore production

Temporal changes in hydrography affect suspended particulate matter (SPM) composition and distribution in coastal systems, potentially influencing the diets of suspension feeders. Temporal variation in SPM and in the diet of the mussel Perna perna, were investigated using stable isotope analysis. The δ13C and δ15 N ratios of SPM, mussels and macroalgae were determined monthly, with SPM samples collected along a 10 km onshore–offshore transect, over 14 months at Kenton-on-Sea, on the south coast of South Africa. Clear nearshore (0 km) to offshore (10 km) carbon depletion gradients were seen in SPM during all months and extended for 50 km offshore on one occasion. Carbon enrichment of coastal SPM in winter (June–August 2004 and May 2005) indicated temporal changes in the nearshore detrital pool, presumably reflecting changes in macroalgal detritus, linked to local changes in coastal hydrography and algal seasonality. Nitrogen patterns were less clear, with SPM enrichment seen between July and October 2004 from 0 to 10 km. Nearshore SPM demonstrated cyclical patterns in carbon over 24-h periods that correlated closely with tidal cycles and mussel carbon signatures, sampled monthly, demonstrated fluctuations that could not be correlated to seasonal or monthly changes in SPM. Macroalgae showed extreme variability in isotopic signatures, with no discernable patterns. IsoSource mixing models indicated over 50% reliance of mussel tissue on nearshore carbon, highlighting the importance of nearshore SPM in mussel diet. Overall, carbon variation in SPM at both large and small temporal scales can be related to hydrographic processes, but is masked in mussels by long-term isotope integration

Contrasting Patterns of Transposable Element Insertions in Drosophila Heat-Shock Promoters

The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila

An AFM study of solid-phase bilayers of unsaturated PC lipids and the lateral distribution of the transmembrane model peptide WALP23 in these bilayers

An altered lipid packing can have a large influence on the properties of the membrane and the lateral distribution of proteins and/or peptides that are associated with the bilayer. Here, it is shown by contact-mode atomic force microscopy that the surface topography of solid-phase bilayers of PC lipids with an unsaturated cis bond in their acyl chains shows surfaces with a large number of line-type packing defects, in contrast to the much smoother surfaces observed for saturated PC lipids. Di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC (POPC) were used. Next, the influence of an altered lipid environment on the lateral distribution of the single α-helical model peptide WALP23 was studied by incorporating the peptide in the bilayers of di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC unsaturated lipids. The presence of WALP23 leads to an increase in the number of packing defects but does not lead to the formation of the striated domains that were previously observed in bilayers of saturated PC lipids and WALP. This is ascribed to the less efficient lateral lipid packing of the unsaturated lipids, while the increase in packing defects is probably an indirect effect of the peptide. Finally, the fact that an altered lipid packing affects the distribution of WALP23 is also confirmed in an additional experiment where the solvent TFE (2,2,2-trifluorethanol) is added to bilayers of di-16:0-PC/WALP23. At 3.5 vol% TFE, the previous striated ordering of the peptide is abolished and replaced by loose lines

Motives and comprehension in a public goods game with induced emotions

This study analyses the sensitivity of public goods contributions through the lens of psychological motives. We report the results of a public goods experiment in which subjects were induced with the motives of care and anger through autobiographical recall. Subjects' preferences, beliefs, and perceptions under each motive are compared with those of subjects experiencing a neutral autobiographical recall control condition. We find, but only for those subjects with the highest comprehension of the game, that care elicits significantly higher contributions than anger, with the control treatment in between. This positive influence of the care motive on unconditional giving is accounted for partly by preferences for giving and partly by the beliefs concerning greater contributions by others. Anger also affects attention to own and other's payoffs (using mouse tracking) and perceptions of the game's incentive structure (cooperative or competitive)

Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors

Activation Mobilizes the Cholesterol in the Late Endosomes-Lysosomes of Niemann Pick Type C Cells

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3–4 hours, ∼4–5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease

Effective transcription factor binding site prediction using a combination of optimization, a genetic algorithm and discriminant analysis to capture distant interactions

<p>Abstract</p> <p>Background</p> <p>Reliable transcription factor binding site (TFBS) prediction methods are essential for computer annotation of large amount of genome sequence data. However, current methods to predict TFBSs are hampered by the high false-positive rates that occur when only sequence conservation at the core binding-sites is considered.</p> <p>Results</p> <p>To improve this situation, we have quantified the performance of several Position Weight Matrix (PWM) algorithms, using exhaustive approaches to find their optimal length and position. We applied these approaches to bio-medically important TFBSs involved in the regulation of cell growth and proliferation as well as in inflammatory, immune, and antiviral responses (NF-κB, ISGF3, IRF1, STAT1), obesity and lipid metabolism (PPAR, SREBP, HNF4), regulation of the steroidogenic (SF-1) and cell cycle (E2F) genes expression. We have also gained extra specificity using a method, entitled SiteGA, which takes into account structural interactions within TFBS core and flanking regions, using a genetic algorithm (GA) with a discriminant function of locally positioned dinucleotide (LPD) frequencies.</p> <p>To ensure a higher confidence in our approach, we applied resampling-jackknife and bootstrap tests for the comparison, it appears that, optimized PWM and SiteGA have shown similar recognition performances. Then we applied SiteGA and optimized PWMs (both separately and together) to sequences in the Eukaryotic Promoter Database (EPD). The resulting SiteGA recognition models can now be used to search sequences for BSs using the web tool, SiteGA.</p> <p>Analysis of dependencies between close and distant LPDs revealed by SiteGA models has shown that the most significant correlations are between close LPDs, and are generally located in the core (footprint) region. A greater number of less significant correlations are mainly between distant LPDs, which spanned both core and flanking regions. When SiteGA and optimized PWM models were applied together, this substantially reduced false positives at least at higher stringencies.</p> <p>Conclusion</p> <p>Based on this analysis, SiteGA adds substantial specificity even to optimized PWMs and may be considered for large-scale genome analysis. It adds to the range of techniques available for TFBS prediction, and EPD analysis has led to a list of genes which appear to be regulated by the above TFs.</p

Gene Expression Analysis of Forskolin Treated Basilar Papillae Identifies MicroRNA181a as a Mediator of Proliferation

Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.Gene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells