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  • Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. 23689370

    Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.
    Document Type:
    Reference
    Product Catalog Number:
    09-838
    Product Catalog Name:
    Anti-Histone H3.3 Antibody

  • Increase in ghrelin levels after weight loss in obese Zucker rats is prevented by gastric banding. 18049841

    BACKGROUND: Gastric banding is thought to decrease appetite in addition to the mechanical effects of food restriction, although this has been difficult to demonstrate in human studies. Our aim was to investigate the changes in orexigenic signals in the obese Zucker rat after gastric banding. METHODS: Obese Zucker rats (fa/fa) were submitted to gastric banding (GBP), sham gastric banding fed ad libitum (sham), or sham operation with food restriction, pair-fed to the gastric banding group (sham-PF). Lean Zucker rats (fa/+) were used as additional controls. Body weight and food intake were daily recorded for 21 days after surgery when epididymal fat was weighed and fasting ghrelin and hypothalamic NPY mRNA expression were measured. RESULTS: Gastric banding in obese Zucker rats resulted in a significant decrease of cumulative body weight gain and food intake. Furthermore, gastric banded rats were leaner than Sham-PF, as expressed by a significantly lower epididymal fat weight. Ghrelin levels of gastric banded rats were not increased when compared to sham-operated animals fed ad libitum and were significantly lower than the levels of weight matched sham-PF rats (1116.9 +/- 103.3 g GBP vs 963.2 +/- 54.3 g sham, 3,079.5 +/- 221.6 sham-PF and 2,969.9 +/- 150.9 g lean rats, p 0.001); hypothalamic NPY mRNA expression was not increased in GBP when compared to sham-operated rats. CONCLUSION: In obese Zucker rats, GBP prevents the increase in orexigenic signals that occur during caloric deprivation. Our data support the hypothesis that sustained weight loss observed after gastric banding does not depend solely on food restriction.
    Document Type:
    Reference
    Product Catalog Number:
    GHRT-89HK
    Product Catalog Name:
    Human Ghrelin (TOTAL) RIA

  • IMPACT OF DIFFERENT VEHICLES FOR LASER-ASSISTED DRUG PERMEATION VIA SKIN: FULL-SURFACE VERSUS FRACTIONAL ABLATION. 24022680

    PURPOSE:
    This study aimed to assess impact of different vehicles for laser-assisted skin drug delivery. We also tried to uncover the mechanisms by which different vehicles affect laser-aided skin permeation.
    METHODS:
    Full-surface ablative (conventional) and fractional lasers were used to irradiate nude mouse skin. Imiquimod and 5-aminolevulinic acid (ALA) were used as lipophilic and hydrophilic permeants. Vehicles employed included water with 40% polyethylene glycol 400 (PEG 400), propylene glycol (PG), and ethanol. Lipid nanoparticles were also utilized as carriers.
    RESULTS:
    In vitro permeation profiles showed improvement in imiquimod flux with conventional laser (2.5 J/cm2) producing a 12-, 9-, and 5-fold increase when loading imiquimod in 40% PEG400, PG, and ethanol, respectively, as compared with intact skin. Nanoparticulate delivery by laser produced a 6-fold enhancement in permeation. Fractional laser produced less enhancement of imiquimod delivery than conventional laser. Laser exposure increased follicular imiquimod accumulation from 0.80 to 5.81 μg/cm2. ALA permeation from aqueous buffer, PEG 400, and PG with conventional laser treatment was 641-, 445-, and 104-fold superior to passive control. In vivo skin deposition of topically applied ALA examined by confocal microscopy indicated the same trend as the in vitro experiment, with aqueous buffer showing the greatest proporphyllin IX signaling. Diffusion of cosolvent molecules into ablated skin and drug partitioning from vehicle to skin are two predominant factors controlling laser-assisted delivery. In contrast to conventional laser, lateral drug diffusion was anticipated for fractional laser.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Spatio-temporal dynamics, differentiation and viability of human neural stem cells after implantation into neonatal rat brain. 21707793

    Neural stem cells (NSCs) have attracted major research interest due to their potential use in cell replacement therapy. In patients, human cells are the preferred choice, one source of human NSCs being the brain of fetuses. The aims of the present study were to explore the long-term differentiation, mobility and viability of NSCs derived from the human fetal striatum in response to intracerebral implantation. To investigate long-term spatio-temporal and functional dynamics of grafts in vivo by magnetic resonance imaging, these cells were labeled with superparamagnetic iron oxide (SPIO) nanoparticles prior to implantation. SPIO-labeling of human NSCs left the quantitative profile of the proliferation, cell composition and differentiation capacity of the cells in vitro unaltered. Also after transplantation, the phenotypes after long-term cell differentiation were not significantly different from naïve cells. Upon transplantation, we detected a hypointensity corresponding to the striatal graft location in all animals and persisting for at least 4 months. The hypointense signal appeared visually similar both in location and in volume over time. However, quantitative volumetric analysis showed that the detectable, apparent graft volume decreased significantly from 3 to 16 weeks. Finally, the human NSCs were not proliferating after implantation, indicating lack of tumor formation. These cells are thus a promising candidate for translationally relevant investigations for stem cell-based regenerative therapies.© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Transcription factor ATF2 regulation by the JNK signal transduction pathway. 7824938

    Treatment of cells with pro-inflammatory cytokines or ultraviolet radiation causes activation of the c-Jun NH2-terminal protein kinase (JNK). Activating transcription factor-2 (ATF2) was found to be a target of the JNK signal transduction pathway. ATF2 was phosphorylated by JNK on two closely spaced threonine residues within the NH2-terminal activation domain. The replacement of these phosphorylation sites with alanine inhibited the transcriptional activity of ATF2. These mutations also inhibited ATF2-stimulated gene expression mediated by the retinoblastoma (Rb) tumor suppressor and the adenovirus early region 1A (E1A) oncoprotein. Furthermore, expression of dominant-negative JNK inhibited ATF2 transcriptional activity. Together, these data demonstrate a role for the JNK signal transduction pathway in transcriptional responses mediated by ATF2.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple