Silibinin inhibits osteoclast differentiation mediated by TNF family members

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.     

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.     

Decoloration of chitosan by UV irradiation

Decoloration of chitosan by UV irradiation, which was used to replace a bleaching step during chitosan preparation, was evaluated under four separate treatments (effect of irradiation time, chitosan/water ratio, stirring speed, and UV light source). The optimal decoloration condition was defined as that producing white chitosan with higher viscosity. Decoloration of chitosan could be achieved effectively using a UV-C light by stirring unbleached chitosan in water (1:8, w/v) for 5 min at 120 rpm. UV irradiation applied under the optimal conditions could be used to produce chitosan with desirable white color (L^* = 76.95, a^* = −0.37, and b^* = 14.04) and high viscosity (1301.7 mPa s at 0.5% w/v in 1.0% v/v acetic acid).     

Depressant Effect of Mitochondrial Respiratory Complex Inhibitors on Proteasome Inhibitor-Induced Mitochondrial Dysfunction and Cell Death in PC12 Cells

The addition of rotenone (inhibitor of respiratory complex I), 3-nitropropionic acid (complex II inhibitor), harmine (inhibitor of complexes I and II) and cyclosporin A (CsA, an inhibitor of the mitochondrial permeability transition) reduced the nuclear damage, loss in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH in differentiated PC12 cells treated with MG132, a proteasome inhibitor. Meanwhile, rotenone, 3-nitropropionic acid and harmine did not affect the inhibitory effect of CsA or trifluoperazine (an inhibitor of the mitochondrial permeability transition and calmodulin antagonist) on the cytotoxicity of MG132. The results suggest that proteasome inhibition-induced mitochondrial dysfunction and cell injury may be attenuated by the inhibitions of respiratory chain complex I and II. The cytoprotective effect of the mitochondrial permeability transition prevention not appears to be modulated by respiratory complex inhibition.     

Mammalian cell cultivation on serum-coated microcarriers

Summary The cell culture on serum-coated microcarriers yielded higher efficiency of cell attachment to microcarriers and more favorable initial cell distribution on microcarriers than on the conventional microcarriers. By employing serum-coated microcarriers, the maximum cell density was increased by 46% in low serum medium and by 30% in 10% (v/v) serum-supplemented medium. Serum coating of microcarriers could provide cell attachment factors and may replace costly attachment factors supplemented in low serum medium and serum-free medium.     

Predicting permanent and transient protein–protein interfaces

Protein–protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. © Proteins 2013. © 2012 Wiley Periodicals, Inc.