Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative <i>in vitro</i> studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin

The Protective Effect of Esculentoside A on Experimental Acute Liver Injury in Mice

<div><p>Inflammatory response and oxidative stress are considered to play an important role in the development of acute liver injury induced by carbon tetrachloride (CCl₄) and galactosamine (GalN)/lipopolysaccharides (LPS). Esculentoside A (EsA), isolated from the Chinese herb phytolacca esculenta, has the effect of modulating immune response, cell proliferation and apoptosis as well as anti-inflammatory effects. The present study is to evaluate the protective effect of EsA on CCl₄ and GalN/LPS-induced acute liver injury. In vitro, CCK-8 assays showed that EsA had no cytotoxicity, while it significantly reduced levels of TNF-α and cell death rate challenged by CCl₄. Moreover, EsA treatment up-regulated PPAR-γ expression of LO2 cells and reduced levels of reactive oxygen species (ROS) challenged by CCl₄. In vivo, EsA prevented mice from CCl₄-induced liver histopathological damage. In addition, levels of AST and ALT were significantly decreased by EsA treatment. Furthermore, the mice treated with EsA had a lower level of TNF-α, Interleukin (IL)-1β and IL-6 in mRNA expression. EsA prevented MDA release and increased GSH-Px activity in liver tissues. Immunohistochemical staining showed that over-expression of F4/80 and CD11b were markedly inhibited by EsA. The western bolt results showed that EsA significantly inhibited CCl₄-induced phosphonated IkBalpha (P-IκB) and ERK. Furthermore, EsA treatment also alleviated GalN/LPS-induced acute liver injury on liver enzyme and histopathological damage. Unfortunately, our results exhibited that EsA had no effects on CCl₄-induced hepatocyte apoptosis which were showed by TUNEL staining and Bax, Caspase-3 and cleaved Caspase-3 expression. Our results proved that EsA treatment attenuated CCl₄ and GalN/LPS-induced acute liver injury in mice and its protective effects might be involved in inhibiting inflammatory response and oxidative stress, but not apoptosis with its underlying mechanism associated with PPAR-γ, NF-κB and ERK signal pathways.</p></div

EsA protected against CCl₄-induced histopathological damage and hepatic dysfunction.

<p>Hematoxylin and eosin staining (A Magnification, 200×) showed that livers in Injury group exhibited more ballooned hepatocytes than those in Control group and EsA group, and symptoms of those histopathological damage were significantly alleviated by EsA treatment (n = 6). Photographs of livers were taken 12 hours post-CCl₄ injection, livers in Injury group turned white (B). Levels of AST and ALT increased obviously after CCl₄ challenge. However, AST and ALT levels did not markedly increase in mice treated with EsA alone, and AST and ALT levels were significantly decreased with EsA treatment. (n = 6 C). The values presented are the means ± standard error of the mean. ^##P<0.01 versus the Control group. *P<0.05, **P<0.01 versus the Injury group.</p

The underlying mechanism of EsA against CCl₄-induced acute liver injury in mice.

<p>The activity of ERK (A) and IκB (B) were determined by western blot. Relative protein levels were quantified by densitometry and expressed as optical density ratio. The values presented are the means ± standard error of the mean (n = 6). ^## P<0.01 versus the Control group. **P<0.01 versus the Injury group.</p

EsA protected against GalN/LPS-induced histopathological damage and hepatic dysfunction.

<p>Hematoxylin and eosin staining (A Magnification, 200×) showed that livers in Injury group exhibited more inflammatory cells than those in Control group and EsA group, which were significantly alleviated by treatment of EsA (n = 6). Liver photographs were taken 12 hours post-GalN/LPS administration, and livers in Injury group turned white (B). Levels of AST and ALT increased obviously after GalN/LPS challenge, and which were significantly decreased with EsA treatment (n = 6 C). The values presented are the means ± standard error of the mean. ^##P<0.01 versus the Control group. *P<0.05, **P<0.01 versus the Injury group.</p

Effects of EsA on CCl₄-induced liver oxidative stress.

<p>EsA treatment significantly decreased levels of MDA (A) and increased the activity of GSH-Px (B) compared with the Injury group. The values presented are the means ± standard error of the mean (n = 6). ^##P<0.01 versus the Control group. *P<0.05 versus the Injury group.</p

Effects of EsA on CCl₄-induced liver inflammation.

<p>mRNA expression of TNF-a, IL-1β and IL-6 (A) and Immunohistochemical staining of F4/80 and CD11b cells (B) accumulating in liver tissues were determined at 12 hours post CCl₄-induced acute liver injury (Magnification, 200×). The values presented are the means ± standard error of the mean (n = 6). ^##P<0.01 versus the Control group. *P<0.05, **P<0.01 versus the Injury group.</p

The molecular structure of Esculentoside A (EsA).

<p>The molecular structure of Esculentoside A (EsA).</p

Effects of EsA on CCl₄-induced LO₂ cell injury and PPAR-γ expression.

<p>The treatment effects of EsA and protein expression of PPAR-γ were measured using western blot (A). Levels of ROS in LO2 cells challenged by CCl₄ were shown (B Magnification, 200×). The mRNA expression of PPAR-γ was measured using quantitative real-time PCR (C). The values presented are the means ± standard error of the mean (n = 5). *P<0.05, **P<0.01.</p

Effects of EsA on cell apoptosis at 12 hours post-CCl₄ injection.

<p>Liver tissues sections were stained with TUNEL method (Magnification, ×200). There were no obvious difference for rates of positive TUNEL stained cells between the Injury and Injury+EsA groups (A). The activity of Bax, Caspase-3 and cleaved Caspase-3 were determined by western blot. Relative protein levels were quantified by densitometry and expressed as optical density ratio (B). The values presented are the means ± standard error of the mean (n = 6). ^#P<0.05, ^##P<0.01 versus the Control group.</p