To study the effects and mechanisms of melatonin (MT) on immune responses in mice of different months. METHODS: Thymocyte proliferation and IL-2 activity were assayed by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) and activated mouse splenocyte proliferation methods, respectively; cAMP and methionine-enkephalin (met-Enk) level was determined by competitive protein binding assay and radioimmunoassay, respectively. RESULTS: The function of lymphocytes, obtained from BALB/c mice aged 6 and 11 months were decreased, which was restored by melatonin at the dose of 5 mg/kg or 30 mg/kg. In vitro, proliferation of lymphocytes in 11-month-old mice was decreased and cAMP level was increased. Melatonin (0.1nmol/L or 1mmol/L) had negative regulation to this. Forskolin (10 mmol/L) enhanced the cAMP level of lymphocytes in 2-and 11-month-old mice (P<0.01), which was antagonized partially by melatonin and this effect of melatonin was also abolished by pertussis toxin (1mg/L) completely. Melatonin (1mmol/L and 0.1nmol/L) increased the content of met-Enk of lymphocytes in 2-and 11-month-old mice, respectively (P<0.01), which was blocked by nifedipine (1 mmol/L). CONCLUSION: Melatonin exerted an effect on immune responses in mice of different months, which might be mediated by G protein-AC-cAMP signal transduction pathway and regulation of met-Enk level.

To investigate the profile of endostatin on adjuvant arthritis (AA) and angiogenesis blockade in synovitis. METHODS: The model of rat AA was induced by injection of intradermal complete Freund's adjuvant (CFA). Hind paw volume of rat was measured by volume meter and the activities of interleukin-1 (IL-1) and IL-2 were measured by the assay of thymocytes proliferation. IL-1β and tumor necrosis factor-α (TNF-α) produced by synoviocytes was estimated with radioimmunoassay. The number of new blood vessels in knee joint synovium was counted under microscope by hematoxylin and eosin (HE) staining. RESULTS: The secondary inflammation of AA rats appeared on the 10th day after injection of CFA. The therapeutic administration of endostatin (0.1, 0.5, and 2.5mg·kg-1·d-1, sc, ×7d) was given from that time (d 10). It was found that endostatin significantly inhibited the secondary paw swelling and the number of new blood vessels in the synovium of AA rats. Endostatin significantly decreased the production of IL-1 derived from both peritoneal macrophages and synoviocytes and IL-2 from splenocytes, especially at the dose of 2.5mg/kg. This effect of endostatin also was seen on TNF-α produced by synoviocytes. CONCLUSION: The recombinant human endostatin had an inhibitory effect on rat AA, which was related to its anti-angiogenesis and inhibition of proinflammatory cytokines.

Endoplasmic reticulum-associated degradation (ERAD)is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosincontaining protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of olyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3, gp78 consistently enhances p97/VCP-CD3 binding and facilitates CD3 degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3, and abrogates VCP-CD3 interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3 degradation and leads to accumulation of polyubiquitinated CD3, suggesting a failure in delivering ubiquitinated CD3 for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.