No abstract

CCAAT/enhancer-binding protein ␣ (C/EBP␣) is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBP␣ is known to induce granulopoiesis while suppressing monocyte differentiation, it is unclear how C/EBP␣ regulates this cell fate choice at the mechanistic level. Here we report that inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBP␣. This inhibition is mediated by extracellular signal-regulated kinases 1 and/or 2 (ERK1/2), which interact with C/EBP␣ through an FXFP docking site and phosphorylate serine 21. As a consequence of C/EBP␣ phosphorylation, induction of granulocyte differentiation by C/EBP␣ or retinoic acid is inhibited. Our analysis of C/EBP␣ by fluorescent resonance energy transfer revealed that phosphorylation induces conformational changes in C/EBP␣, increasing the distance between the amino termini of C/EBP␣ dimers. Thus, myeloid development is partly regulated by an ERK1/2-mediated change in the conformation of C/EBP␣ that favors monocyte differentiation by blocking granulopoiesis.

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/ enhancer-binding protein ␣ (C/EBP␣) to the mouse major ␣-satellite repetitive DNA sequesters C/EBP␣ in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBP␣. Functional sequestration of C/EBP␣ was demonstrated by experimentally reducing C/EBP␣ binding to the major ␣-satellite DNA, which elevated the concentration of C/EBP␣ in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBP␣ binding to ␣-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBP␣ from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBP␣ that reduces binding to ␣-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of ␣-satellite DNA binding coincided with an elevation in the binding of C/EBP␣ to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBP␣ to this highly repetitive DNA reduced the amount of C/EBP␣ available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

The established in vitro tool used for testing the absorption and penetration of chemicals through skin in pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin permeation platform and validate it rigorously against the Franz cell by comparing the transport of 3 model chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and comparable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip decreases the effect of unstirred water layers that can occur in static Franz cells. The validation reported herein sets the stage for efficient skin permeation and toxicity screening and further development of microfluidic skin-on-chip devices.

Articles you may be interested inSurface and grain boundary scattering in nanometric Cu thin films: A quantitative analysis including twin boundaries J.

No abstract

We present an experimental approach to systematically produce nanostructures with various grain sizes and twin densities in the Ni-Co binary system. Using electrodeposition with various applied current densities and organic additive contents in the deposition bath, we synthesize nanostructured fcc and hcp solid solutions with a range of compositions. Due to the low stacking fault energy (SFE) of these alloys, growth twins are readily formed during deposition, and by adjusting the deposition conditions, a range of twin boundary densities is possible. The resulting nanostructured alloys cannot be described by a single characteristic length scale, but instead must be characterized in terms of (1) a true grain size pertaining to general high-angle grain boundaries and (2) an effective grain size that incorporates twin boundaries. Analysis of Hall-Petch strength scaling for these materials is complicated by their dual length scales, but the hardness trends found in Ni-80Co are found to be roughly in line with those seen in pure nanocrystalline nickel.

Hepatocytes isolated from male F344 rats were exposed to elevated temperatures (40 degrees C to 45 degrees C), and the incorporation of [35S]-L-methionine into proteins was measured from fluorograms of two-dimensional polyacrylamide gels. The synthesis of two proteins was induced by temperatures of 40 degrees C to 42.5 degrees C; however, 45 degrees C inhibited the synthesis of all proteins. Based on their apparent molecular weights and pI values and their recognition by a monoclonal antibody to the HSP70 gene family, the two proteins induced by hyperthermia were found to be the highly heat-inducible hsp70 and the constitutive hsc70. Because a heat shock of 42.5 degrees C for 30 minutes had very little effect on cell viability and induced the synthesis of hsp70 and hsc70, this heat shock was used to study the effect of age on the ability of hepatocytes to respond to the stress of hyperthermia. Neither hepatocytes isolated from young adult (5-7 months) nor old (25-27 months) rats synthesized detectable amounts of hsp70 when incubated at 37 degrees C. However, heat shock induced the synthesis of both hsp70 and hsc70 in hepatocytes isolated from young adult and old rats. The induction of hsp70 synthesis was significantly lower (37%) for hepatocytes isolated from old rats compared to hepatocytes isolated from young adult rats. However, neither the basal level nor the induced level of hsc70 synthesis changed significantly with age. Thus, aging resulted in a decrease in the ability of hepatocytes to synthesize hsp70 in response to hyperthermia; this effect, however, was specific for hsp70.