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In this study, we found that catechins found in green tea (EGCG, EGC, and EC) differentially interfere with the IL-1β signaling pathway which regulates the expression of pro-inflammatory mediators (IL-6 and IL-8) and Cox-2 in primary human rheumatoid arthritis synovial fibroblasts (RASFs). EGCG and EGC inhibited IL-6, IL-8, and MMP-2 production and selectively inhibited Cox-2 expression. EC did not exhibit any inhibitory effects. When we looked at the expression of key signaling proteins in the IL-1β signaling pathway, we found all the tested catechins could inhibit TAK-1 activity. Therefore, the consumption of green tea offers an overall anti-inflammatory effect. Molecular docking analysis confirms that EGCG, EGC, and EC all occupy the active site of the TAK1 kinase domain. However, EGCG occupies the majority of the TAK1 active site. In addition to TAK1 inhibition, EGCG can also inhibit P38 and nuclear NF-κB expression whereas EC and EGC were not effective inhibitors. Our findings suggest one of the main health benefits associated with the consumption of green tea are due to the activity of EGCG and EGC which are both present at higher amounts. Although EGCG is the most effective catechin at inhibiting downstream inflammatory signaling, its effectiveness could be hindered by the presence of EC. Therefore, varying EC content in green tea may reduce the anti-inflammatory effects of other potential catechins in green tea.

Background/Purpose Transforming growth factor β activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), or toll-like receptor signaling. We examined the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA). Methods The effect of TAK1, IRAK-1, or TRAF6 inhibition was evaluated in IL-1β-induced human RA synovial fibroblasts (RA-FLS). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RA-FLS. The efficacy of epigallocatechin-3-gallate (EGCG), a potent anti-inflammatory molecule, in regulating these processes in RA-FLS was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were validated using a rat adjuvant-induced arthritis (AIA) model. Results Inhibition of TAK1, not IRAK-1 or TRAF6, completely abrogates IL-1β-induced IL-6 and IL-8 synthesis in RA-FLS. EGCG inhibits TAK1 phosphorylation at Thr184/187 and occupies the C174 position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibits K63-autoubiquitination of TRAF6, a post-translational modification essential for TAK1 autophosphorylation, by forming stable hydrogen-bond at the K124 position on TRAF6. Furthermore, EGCG enhances associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses on the joint homogenates from rat AIA show a significant increase in K48-linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to the naïve group. Administration of EGCG (50 mg/kg/day) for 10 days ameliorates AIA by reducing TAK1 phosphorylation and K48 polyubiquitination. Conclusions This study provides rationale for targeting TAK1 for RA treatment by EGCG.

Aim:Identification and determination of sex of unknown human skeletal remains has been one of the most challenging tasks for forensic dentistry. The purpose of this study was to determine the gender from the analysis of mental foramen on panoramic radiographs in a north Indian population.Materials and Methods:One hundred radiographs were selected for the analysis of mental foramen. Tangents were drawn to the superior and inferior borders of the foramen and perpendiculars were drawn from the tangents to the lower border of the mandible (S-L and I-L). The data obtained were tabulated and subjected to statistical analysis.Results:The average values of S-L and I-L were significantly higher in males than in females, while the distances for the right and left sides of an individual were almost similar in both the male and the females group, and the results were non-significant.Conclusion:The distances from the mental foramen to the lower border of the mandible exhibit sexual dimorphism in the north Indian population.

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein.The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but -mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration-and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from R-helical to -sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.