AB4140 Sigma-AldrichAnti-MOP3 Antibody

Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA.Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α.Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions.We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.

Fully mature enamel is about 98% mineral by weight. While mineral crystals appear very early during its formative phase, the newly secreted enamel is a soft gel-like matrix containing several enamel matrix proteins of which the most abundant is amelogenin (Amelx). Histological analysis of mineralized dental enamel reveals markings called cross-striations associated with daily increments of enamel formation, as evidenced by injections of labeling dyes at known time intervals. The daily incremental growth of enamel has led to the hypothesis that the circadian clock might be involved in the regulation of enamel development. To identify daily rhythms of clock genes and Amelx, we subjected murine ameloblast cells to serum synchronization to analyze the expression of the circadian transcription factors Per2 and Bmal1 by real-time PCR. Results indicate that these key genetic regulators of the circadian clock are expressed in synchronized murine ameloblast cell cultures and that their expression profile follows a circadian pattern with acrophase and bathyphase for both gene transcripts in antiphase. Immunohistological analysis confirms the protein expression of Bmal and Cry in enamel cells. Amelx expression in 2-day postnatal mouse molars dissected every 4 hours for a duration of 48 hours oscillated with an approximately 24-hour period, with a significant approximately 2-fold decrease in expression during the dark period compared to the light period. The expression of genes involved in bicarbonate production (Car2) and transport (Slc4a4), as well as in enamel matrix endocytosis (Lamp1), was greater during the dark period, indicating that ameloblasts express these proteins when Amelx expression is at the nadir. The human and mouse Amelx genes each contain a single nonconserved E-box element within 10 kb upstream of their respective transcription start sites. We also found that within 2 kb of the transcription start site of the human NFYA gene, which encodes a positive regulator of amelogenin, there is an E-box element that is conserved in rodents and other mammals. Moreover, we found that Nfya expression in serum-synchronized murine ameloblasts oscillated with a strong 24-hour rhythm. Taken together, our data support the hypothesis that the circadian clock temporally regulates enamel development.