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Several polymers have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a direct in vivo comparison of delivery systems based on Pluronic P85, Myrj 52 and chitosan-4-thiobutylamidine (Ch-TBA) in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. Furthermore, the postulated low molecular mass P-gp inhibitors 6-mercaptopurine and reduced glutathione (GSH) were evaluated in vitro and in vivo. In vitro, the permeation enhancing effect of 6-mercaptopurine, GSH, Pluronic P85, Myrj 52, and the combination of Ch-TBA with GSH was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type diffusion chambers. In comparison to buffer only, Rho-123 transport in presence of 100mum 6-mercaptopurine, 0.5%(w/v) GSH, 0.5%(w/v) Pluronic P85, 0.5%(w/v) Myrj 52 and the combination of 0.5%(w/v) Ch-TBA/ 0.5%(w/v) GSH, was 2.1, 1.6, 1.9, 1.8, 3.0-fold improved, respectively. In vivo in rat, enteric-coated tablets based on Pluronic P85, Myrj 52 or Ch-TBA/GSH increased the area under the plasma concentration time curve (AUC(0-12)) of Rho-123 1.6-fold, 2.4-fold, 4.3-fold, respectively, in comparison to control only. Contrariwise, the low molecular mass excipients 6-mercaptopurine and GSH showed no significant effect in vivo at all. This in vivo study showed that polymeric P-gp inhibitors and especially the delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.

Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the α-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in PP, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general.

The mapping of expression quantitative trait loci (eQTLs) has emerged as an important tool for linking genetic variation to changes in gene regulation. However, it remains difficult to identify the causal variants underlying eQTLs, and little is known about the regulatory mechanisms by which they act. Here we show that genetic variants that modify chromatin accessibility and transcription factor binding are a major mechanism through which genetic variation leads to gene expression differences among humans. We used DNase I sequencing to measure chromatin accessibility in 70 Yoruba lymphoblastoid cell lines, for which genome-wide genotypes and estimates of gene expression levels are also available. We obtained a total of 2.7 billion uniquely mapped DNase I-sequencing (DNase-seq) reads, which allowed us to produce genome-wide maps of chromatin accessibility for each individual. We identified 8,902 locations at which the DNase-seq read depth correlated significantly with genotype at a nearby single nucleotide polymorphism or insertion/deletion (false discovery rate = 10%). We call such variants 'DNase I sensitivity quantitative trait loci'(dsQTLs). We found that dsQTLs are strongly enriched within inferred transcription factor binding sites and are frequently associated with allele-specific changes in transcription factor binding. A substantial fraction (16%) of dsQTLs are also associated with variation in the expression levels of nearby genes (that is, these loci are also classified as eQTLs). Conversely, we estimate that as many as 55% of eQTL single nucleotide polymorphisms are also dsQTLs. Our observations indicate that dsQTLs are highly abundant in the human genome and are likely to be important contributors to phenotypic variation.

Despite resveratrol's well-documented health benefits, its mechanism of action remains controversial. In particular, the direct molecular target of resveratrol has been elusive. Park et al. now show that resveratrol directly inhibits cAMP-dependent phosphodiesterases, triggering a cascade of events that converge on the important energy-sensing metabolic regulators AMPK, SIRT1, and PGC-1α.

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. Here, we demonstrate that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this "dormant" TF binding signature may represent a molecular footprint of these cells' developmental lineage.

Similar to hematopoietic stem cells, memory lymphocytes self-renew, while their clonally expanded effector progeny differentiate to fight infection and tumors. Recently, Muranski et al.(2011) report in Immunity that a subset of Th17 effector cells function as memory cells and retain stem cell properties.

BACKGROUND: Epithelial tissues undergo extensive collective movements during morphogenesis, repair, and renewal. Collective epithelial cell migration requires the intercellular coordination of cell-cell adhesions and the establishment of anterior-posterior polarity, while maintaining apical-basal polarity, but how this is achieved at the molecular level is not well understood. RESULTS: Using an RNA interference-based screen to identify Rho family GTPase regulators required for the collective migration of human bronchial epithelial cells, we identified myosin-IXA (gene name: Myo9a). Depletion of myosin-IXA, a RhoGAP and actin motor protein, in collectively migrating cells led to altered organization of the actin cytoskeleton and tension-dependent disruption of cell-cell adhesions, followed by an inability to form new adhesions resulting in cell scattering. Closer examination revealed that myosin-IXA is required during the formation of junction-associated actin bundles soon after cell-cell contact. Structure-function analysis of myosin-IXA revealed that the motor domain is necessary and sufficient for binding to actin filaments, whereas expression of the RhoGAP domain partially rescued the cell scattering phenotype induced by myosin-IXA depletion. Finally, a fluorescence resonance energy transfer biosensor revealed a significant increase in Rho activity at nascent cell-cell contacts in myosin-IXA depleted cells compared to controls. CONCLUSION: We propose that myosin-IXA locally regulates Rho and the assembly of thin actin bundles associated with nascent cell-cell adhesions and that this is required to sustain the collective migration of epithelial cells.

Despite fluctuations in dietary iron intake and intermittent losses through bleeding, the plasma iron concentrations in humans remain stable at 10-30μM. While most of the iron entering blood plasma comes from recycling, appropriate amount of iron is absorbed from the diet to compensate for losses and maintain nontoxic amounts in stores. Plasma iron concentration and iron distribution are similarly regulated in laboratory rodents. The hepatic peptide hepcidin was identified as the systemic iron-regulatory hormone. In the efferent arc, hepcidin regulates intestinal iron absorption, plasma iron concentrations, and tissue iron distribution by inducing degradation of its receptor, the cellular iron exporter ferroportin. Ferroportin exports iron into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron. In the more complex and less well understood afferent arc, hepatic hepcidin synthesis is transcriptionally regulated by extracellular and intracellular iron concentrations through a molecular complex of bone morphogenetic protein receptors and their iron-specific ligands, modulators and iron sensors. Through as yet undefined pathways, hepcidin is also homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In accordance with the role of hepcidin-mediated iron redistribution in host defense, hepcidin production is regulated by inflammation as well. Increased hepcidin concentrations in plasma are pathogenic in iron-restrictive anemias including anemias associated with inflammation, chronic kidney disease and some cancers. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and ineffective erythropoiesis. Hepcidin, ferroportin and their regulators represent potential targets for the diagnosis and treatment of iron disorders and anemias. This article is part of a Special Issue entitled: Cell Biology of Metals.