Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well‐known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβ3 and αVβ5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), matrix metalloproteinase‐3 (MMP‐3), and MMP‐13 induced by excessive mechanical stress. In addition, the protein expression of IL1‐β and MMP‐13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress‐induced integrin signaling in chondrocytes by using western blotting. The levels of p‐FAK, p‐ERK, p‐JNK, and p‐p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVβ3 and αVβ5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL‐1β, TNF‐α, MMP‐3, and MMP‐13 through phosphorylation of FAK and MAPKs.     

Optical resolution and enantiomeric purity determination of the analgesic cizolirtine

The optical resolution of (±)‐cizolirtine was accomplished with excellent results (>99% ee) by means of crystallization with (+)‐ or (−)‐di‐p‐toluoyltartaric acid. The optical purity of the samples was controlled by three independent methods: ¹H NMR, capillary electrophoresis (CE) (using β‐cyclodextrins as chiral resolving agents), and HPLC (using a glycoproteic column). The use of a rapid analytical technique like ¹H NMR for estimating the relative amounts of each enantiomer, together with the high sensitivity of CE, afforded a convenient strategy for monitoring the entire process leading to enantiopure compounds. Chirality 11:63–69, 1999. © 1999 Wiley‐Liss, Inc.     

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.     

GSK‐3β inhibition protects the rat heart from the lipopolysaccharide‐induced inflammation injury via suppressing FOXO3A activity

Sepsis‐induced cardiac dysfunction represents a main cause of death in intensive care units. Previous studies have indicated that GSK‐3β is involved in the modulation of sepsis. However, the signalling details of GSK‐3β regulation in endotoxin lipopolysaccharide (LPS)‐induced septic myocardial dysfunction are still unclear. Here, based on the rat septic myocardial injury model, we found that LPS could induce GSK‐3β phosphorylation at its active site (Y216) and up‐regulate FOXO3A level in primary cardiomyocytes. The FOXO3A expression was significantly reduced by GSK‐3β inhibitors and further reversed through β‐catenin knock‐down. This pharmacological inhibition of GSK‐3β attenuated the LPS‐induced cell injury via mediating β‐catenin signalling, which could be abolished by FOXO3A activation. In vivo, GSK‐3β suppression consistently improved cardiac function and relieved heart injury induced by LPS. In addition, the increase in inflammatory cytokines in LPS‐induced model was also blocked by inhibition of GSK‐3β, which curbed both ERK and NF‐κB pathways, and suppressed cardiomyocyte apoptosis via activating the AMP‐activated protein kinase (AMPK). Our results demonstrate that GSK‐3β inhibition attenuates myocardial injury induced by endotoxin that mediates the activation of FOXO3A, which suggests a potential target for the therapy of septic cardiac dysfunction.     

Charged cyclodextrin-mediated sample stacking in micellar capillary electrophoresis: A simple method for enhancing the detection sensitivity of hydrophobic compounds

The development of on-line sample stacking techniques for enhancing limits of detection of neutral analytes in micellar capillary electrophoresis (MCE) has recently gained much attention. Utilizing high-conductivity sample matrices to invoke sample stacking is promising, but requires the limited use of sample solubilizing agents such as alcohols in the sample matrix. In this study, we show how simple replacement of the sample solvent (methanol) with a solution of sulfated β-cyclodextrin (sβ-CD) allows a significant increase in the sensitivity of detection of model hydrophobic analytes. This increase in sensitivity is accompanied by significant peak sharpening. Sulfated CDs in the sample matrix allow for effective solubilization of hydrophobic analytes without the use of organic solvents such as methanol. The testing of various sample matrix sβ-CD concentrations for their effect on peak sharpening identified 3 to 5% as optimal for the estrogen standards. The use of a sβ-CD sample matrix allowed for hydrostatic injections (3.5 kPa) of 297 s, compared with 4 s when the analytes were dissolved in methanol. A mechanism explaining the sβ-CD-induced effect involves an analyte transfer mechanism where the sβ-CDs, despite providing anodic mobility to analytes in the sample zone, are able to transfer analytes to trailing separation buffer micelles for “recycling” back into the sample zone without compromising the stacking process. The overall improvement in sensitivity allows detection of estrogens in the parts-per-billion range and stands to improve the utility of MCE as a bioanalytical technique.     

Therapeutic potential of α,β‐thujone through metabolic reprogramming and caspase‐dependent apoptosis in ovarian cancer cells

The therapeutic potential of α,β‐thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,β‐thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,β‐thujone inhibited cancer cell proliferation and induced cell death through caspase‐dependent intrinsic apoptotic pathways. Moreover, α,β‐thujone‐mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,β‐Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,β‐thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,β‐thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress‐associated metabolic reprogramming and caspase‐dependent apoptotic pathways.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanistic target of rapamycin controls homeostasis of adipogenesis

Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.     

β‐Hydroxybutyrate exacerbates lipopolysaccharide/ d‐galactosamine‐induced inflammatory response and hepatocyte apoptosis in mice

β‐Hydroxybutyrate (BHB), one of ketone body, has been traditionally regarded as an alternative carrier of energy, but recent studies found that BHB plays versatile roles in inflammation. It has been previously reported that the level BHB declined in mice with lipopolysaccharide (LPS)/d ‐galactosamine (d ‐Gal)‐induced liver damage, but the pathological significance remains unclear. In the present study, the pathophysiological roles of BHB in LPS/d ‐Gal‐induced hepatic damage has been investigated. The results indicated pretreatment with BHB further enhanced LPS/d ‐Gal‐induced elevation of aspartate aminotransferase and alanine aminotransferase, exacerbated the histological abnormalities and increased the mortality. Pretreatment with BHB upregulated the level of tumor necrosis factor α and interleukin‐6 in plasma, promoted the activities of caspase‐3, caspase‐8, and caspase‐9 and increased the count of terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive cells. In addition, post‐insult supplement with BHB also potentiated LPS/d ‐Gal‐induced apoptotic liver damage. Therefore, BHB might be a detrimental factor in LPS/d ‐Gal‐induced liver injury via enhancing the inflammation and the apoptosis in the liver.     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

Pathophysiological roles of chronic low‐grade inflammation mediators in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common hormonal imbalance disease in reproductive‐aged women. Its basic characteristics are ovulatory dysfunction and ovarian overproduction of androgens that lead to severe symptoms such as insulin resistance, hirsutism, infertility, and acne. Notwithstanding the disease burden, its underlying mechanisms remain unknown, and no causal therapeutic exists. In recent years, further studies showed that inflammation processes are involved in ovulation and play a key role in ovarian follicular dynamics. Visceral adipose tissue can cause inflammatory response and maintenance of the inflammation state in adipocytes by augmented production of inflammatory cytokines, monocyte chemoattractant proteins, and recruitment of the immune cell. Therefore, the PCOS can be related to a low‐grade inflammation state and inflammatory markers. Investigating the inflammatory processes and mediators that contribute to the commencement and development of PCOS can be a critical step for better understanding the pathophysiology of the disease and its treatment through inhibition or control of related pathways. In the present review, we discuss the pathophysiological roles of chronic low‐grade inflammation mediators including inflammasome‐related cytokines, interleukin‐1β (IL‐1β), and IL‐18 in PCOS development.