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Objective To investigate the relationship between the levels of serum glial fibrillary acidic protein (GFAP) ,ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) ,CT findings and outcome in patients with severe traumatic brain injury .Methods En-zyme linked immunosorbent assay (ELISA) was conducted to detect the serum level of GFAP and UCH-L1 in 62 patients with se-vere traumatic brain injury at the time of 12 h and 36 h after the trauma .CT scans of the brain were obtained within 12 h of injury . Outcome was assessed by glasgow outcome scale (GOS) at 6th month .The relationship between GFAP ,UCH-1 ,CT findings and outcome were analyzed .56 healthy persons were selected as control group .Results The concentrations of serum GFAP and UCH-L1 of patients were significantly higher than those of the control group (P< 0 .05) ;GFAP levels were higher in patients with mass lesions than in those with diffuse injury while UCH-L1 levels were higher in patients with diffuse injury (P< 0 .05) ;the levels of GFAP and UCH-L1 of patients with unfavourable outcome(GOS 1 - 3 scores) were significantly higher than those of patients with favourable outcome(GOS 4 - 5 scores) ,and the concentrations of biomarkers were significantly negatively correlated with outcome . Conclusion Serum levels of GFAP and UCH-L1 are good predictors for severity and outcome in severe traumatic brain injury (TBI) .The levels of GFAP and UCH-L1 could reflect different injury pathways which were different in patients with mass lesions and diffuse injury remarkbly .GFAP and UCH-L1 could provide better characterization of subjects for specific types of cellular dam -age than that obtained with CT alone .
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Objective To investigate the effects of the rat serum with chronic renal failure (CRF) on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4) of rat arterial vascular smooth muscle cells(VSMCs) cultured in vitro,and explore the possible mechanism.Methods To establish the rat model of CRF by 5/6 nephrectomy,VSMCs were incubated in the media with the 10％ of CRF serum or control serum in vitro.The mRNA expressions of ubiquitin(Ub),ubiquitin activating enzyme(E1),ubiquitin ligases enzymes (β-transducin repeat containing protein 1,β-TrCP1),p300 and ATF4 in the rat VSMCs were examined by using realtime PCR.Expressions of E1,β-TrCP1,p300 and ATF4 proteins in response to the CRF serum in VSMCs were determined by Western blotting analysis.The enzyme activities of 20S proteasomes in the total protein were examined by using three special fluorogenic peptide substrates.Results The CRF serum significantly promoted the mRNA expressions of Ub,E1,β-TrCP1,p300 and ATF4 in VSMCs in a time dependent manner.Compared with that in control serum group,the mRNA levels of Ub,E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The CRF serum also increased the protein expressions of E1,β-TrCP1 and p300 in a time dependent manner.The expression of ATF4 was decreased,but the difference was not significant (P ＞ 0.05).Compared with that in control serum group,the protein expressions of E1,β-TrCP1,p300 and ATF4 in CRF serum group increased significantly (P ＜ 0.01).The activities of 20S proteasomes in the CRF serum group were significantly increased in a time dependent manner.Compared with that in control serum group,the activities of 20S proteasomes in the CRF serum group increased significantly (P ＜ 0.01).Conclusions The serum of CRF rat can effectively active the ubiquitin-proteasome pathway,but ATF4 ubiquitinylated degradation is blocked.The latter may be associated with increased expression of p300.
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Background Oxidative damage is a major cause of age-related cataracts,and the ubiquitinproteasome system is involved in lens differentiation and development.Ubiquitin carboxy-terminal hydrolase L1 (UCHL1),one of key enzymes of ubiquitin-proteasome system,was discovered to participate in the age related diseases and oxidative stress damage. Objective This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods Lens capsule were collected from 24 patients with age-related cataract(including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery.Five normal lens capsule membranes were obtained from eye bank of Tongji University.Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study.Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology.UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome,and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group.UCHL1 over-expressing cells were then exposed to different concentrations (0.2,0.3,0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer,but significantly different expressing levels were seen among normal lens capsular membrane,cortical cataract and nuclear cataract ( F =13.411,P =0.000),and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P =0.000,P =0.000).No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract ( P =0.164).Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group,illuminating a successful transfection of UCHL1 in SRA01/04 cells.MMT assay revealed that the A570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions UCHL1 has an antioxidative ability,and it might plays an important role in the progress of age-related cataract.
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Ubiquitination on target proteins is the signal of cellular protein degradation.Ubiquitin ligase E3 is one of the key enzymes in ubiquitination,it recognizes a specific substrate protein and recruits an ubiquitin conjugating enzyme E2,mediating the ubquitin transfer from the E2 to the substrate protein.Ubiquitin ligase E3 can be divided into two subfamilies according to their different structure characters and function mechanisms,the HECT(homologous to E6AP C terminus) family and the RING-finger family.Members of the HECT E3 share the common HECT catalytic domain,which can bind to an E2 and load the ubiquitin on themselves before catalyzing the transfer of ubiquitin to the target proteins.While the RING-finger E3 all contain an similar E2 binding domain and a unique substrate binding part,mediating direct ubiquitin transfer from the E2 to the substrate.The most recent progresses in the stuctural and functional studies of these two E3 famlies were summarized.