Ginsenoside Rd, a natural production for attenuating fibrogenesis and inflammation in hepatic fibrosis by regulating the ERRα-mediated P2X7r pathway.

Ginseng, when used as a food and nutritional supplement, has the ability to regulate human immunity. Here, the potential anti-hepatic fibrosis effect of ginsenoside Rd (Rd), one of the protopanaxadiol types of ginsenoside, was investigated. We established a hepatic fibrosis model using intraperitoneal injection of thioacetamide (TAA) for five weeks in mice. In addition, an in vitro model was established by using TGF-ß to activate hepatic stellate cells (HSCs), treated with Rd and an estrogen-related receptor α (ERRα) inhibitor (XCT-790). The ERRα knockdown (shRNA-ERRα) of the primary mouse hepatocytes was used to establish hepatocyte injury by TGF-ß, and they were then incubated in Rd. The Rd significantly alleviated the histopathological changes, and reduced the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The Rd could upregulate the ERRα and downregulate the fibrosis markers in the livers of mice. In TAA-induced mice, the Rd inhibited the purinergic ligand-gated ion channel 7 receptor (P2X7r)-mediated NLRP3 inflammasome activation, consequently reversing the liver inflammatory response. The Rd significantly increased the expression of ERRα and suppressed the extracellular matrix (ECM) in the HSCs or primary hepatocytes. The Rd significantly decreased the P2X7r-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of IL-1ß, IL-23 in the activated HSCs and primary hepatocytes. The Rd could ameliorate the damage of the hepatocytes and further inhibit the entry of IL-1ß and IL-18 into the extracellular matrix. The Rd reduced the inflammatory reaction by regulating the ERRα-P2X7r signaling pathway while suppressing the fibrogenesis, which suggests that the Rd can serve as a novel dietary supplement approach to combat hepatic fibrosis.

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation.

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/µL phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer's disease.

Limited proteolysis via millisecond digestions in protease-modified membranes.

Sequential adsorption of poly(styrene sulfonate) (PSS) and proteases in porous nylon yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm(3)) and small pore diameters in the membrane lead to digestion in <1 s, the low membrane thickness (170 µm) affords control over residence times at the millisecond level to limit digestion. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Moreover, electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) on a large myoglobin proteolytic peptide (8 kDa) provides a resolution of 1-2 amino acids. Under denaturing conditions, limited membrane digestion of bovine serum albumin (BSA) and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kDa) that increase the sequence coverage from 53% (2 s digestion) to 82% (0.05 s digestion). With this approach, we also performed membrane-based limited proteolysis of a large Arabidopsis GTPase, Root Hair Defective 3 (RHD3) and showed suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization.

Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

[Effect of NAD+ against radiation injury and its dose-effect relationship].

OBJECTIVE: To explore the effect of NAD+ against radiation injury and its dose-effect relationship. METHODS: L02 liver cells cultured in RPMI 1640 medium containing 10% fetal calf serum were exposed to X-ray irradiation followed by immediate application of NAD+. The cellular viability was analyzed by MTT assay and the apoptotic cells were detected by TUNEL methods to observe the damages of L02 liver cells induced by X-ray exposure and analyze the dose-effect relationship of NAD+. RESULTS: The viability of L02 liver cells was decreased with increasing dose of X-ray irradiation. The most obvious growth inhibition of L02 cells occurred 24 h after the irradiation. NAD+ significantly increased the cell survival rate after irradiation, and this effect was gradually increased within the concentration range of 100-1000 microg/ml; at higher concentrations, the survival rate of the irradiated L02 cells showed no significant increase. CONCLUSION: NAD+ provides partial protection of the liver cells against radiation injury, and the effect is positively correlated to NAD+ concentration within a certain range.

Identification of p65-associated phosphoproteins by mass spectrometry after on-plate phosphopeptide enrichment using polymer-oxotitanium films.

Stimuli-induced protein phosphorylation plays a vital role in signal transduction and transcriptional activities in eukaryotic cells. This work aims to develop analysis techniques that rapidly detect stimulus-specific intracellular protein phosphorylation and association, with specific emphasis on identifying phosphoproteins associated with p65, a nuclear regulatory factor. The analytical strategy includes immunoprecipitation of the target protein along with its associated proteins, tryptic digestion directly on the antibody beads, on-plate phosphopeptide enrichment for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) to identify phosphopeptides and phosphorylation sites. Enrichment of the phosphopeptides from the tryptic digest occurs on a polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) and consumes as little as 1 microL of digest solution. The Au-P-oxoTi wafers can capture both mono- and multiphosphorylated peptides from model protein digests containing high concentrations of nonphosphopeptides, urea, and salts. When combined with MALDI-MS/MS, the enrichment technique reveals nine phosphopeptides from p65-associated proteins immunoprecipitated from human acute monocytic leukemia (THP-1) cell nuclear extracts. Semiquantitative MALDI-MS shows that the levels of these proteins increase dramatically after treatment with tumor necrosis factor (TNF)-alpha. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.