Objective:To study the mechanism of immune tolerance induce by protal venous injection of donor spleen cells on the dog model of renal transplantation.Methods:The donor spleen cells were injected through the protal vein during operation,one week later,renal transplantation was performed.IL-2 and IL-6 were studied by method of ELISA.Results:Level of IL-2 and IL-6 in protal venous group and cyclosporin group was higher than that of control group.There were no difference between protal venous group and cyclosporin group.Conclusion:Immune tolerance could be produced by protal venous injection of donor spleen cells.

Objective To explore the effect of transforming growth factor β1 (TGF-β1) on epigenetic histone lysine acetylation in the plasminogen activator inhibitor 1 (PAI-1) promoter and transcribe regions in glomerular mesangial cells (GMCs).Methods Chromatin immunoprecipitation assay and real-time quantitative PCR were used to detect Histone3K9 acetylation (H3K9Ac) in the PAI-1 promoter and transcribe regions induced by TGF-β1 and high glucose.Immunoprecipitation was also used to see the cooperation of Smad3,CBP and Sp1 proteins.Results In the four target regions of PAI-1 promoter,TGF-β1 treatment enhanced H3K9Ac at P1,P2 and P3 in GMCs (P ＜ 0.05),but no change was seen in the P4 region which was far from the transcription starting site.TGF-β1 obviously induced H3K9Ac in the T1 transcribe region of PAI-1 instead of T2 (P ＜ 0.05).High glucose increased PAI-1 mRNA expression and H3K9Ac around P1 promoter region (P＜ 0.05).TGF-β1 neutralizing antibody abrogated high glucose-induced H3K9Ac at PAI-1 promoter (P ＜ 0.01).TGF-β1 treatment could recruit Smad3 and CBP protein binding to the PAI-1 promoter regions (P1,P2,P3),and induce their cooperation in GMCs,which were responsing to TGF-β1 associated H3K9Ac.Conclusion TGF-β1 can induce H3K9Ac in the promoter and transcribe regions of PAI-1,promote Smad3 recruition and cooperation with Sp1 and CBP,which are associated with PAI-1 gene's regulation in GMCs.

Objective To investigate the effects of 12-lipoxygenase (12-LO) and angiotensin Ⅱ (Ang Ⅱ) on the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs) p21,p27 and p57 related to cell hypertrophy.Methods Mesangial cells were treated with high glucose for 24 hours and 48 hours respectively.12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and Ang Ⅱ were infused to rats by osmotic mini-pump for 1 week and 2 weeks respectively.Rats fed high fat diet were received low dose streptozotocin (STZ) to make type 2 diabetes (DN).The rats were divided into normal control group,DN group,DN+Ang Ⅱ type 1 receptor blocker (ARB) group or 12-LO inhibitor (CDC) group.DN+ARB rats were treated by losartan for 6 weeks,and DN+CDC rats were treated for 8 weeks.Urine albumin and protein expressions of p21,p27 and p57 were detected by ELISA and Western blotting respectively.Glomeruli injury and expressions of p21 and p27 were detected by PAS staining and immunohistochemistry respectively.Results High glucose increased p21 and p27 protein expression in mesangial cells significantly compared with the relative control (all P ＜ 0.05),but had no effect on p57.Ang Ⅱ increased p27 protein expression in gloneruli significantly (P ＜ 0.05),but had no effect on p21 and p57 protein expression.12(S)-HETE increased both p21 and p27 protein expression in glomeruli significantly (all P ＜ 0.05),but had no effect on p57 protein expression.Blood glucose,kidney/body weight,urinary protein,and glomerular p21 and p27 protein expressions were increased in DN group (all P ＜ 0.05) compared with those in control group,with little change of p57 protein expression (P ＜ 0.05).Moreover,glomerular hypertrophy and extra cellular matrix accumulation were observed in DN group.However,urine protein,kidney/body weight,renal injury,but not blood glucose,were decreased in DN+ARB group and DN+CDC group compared with DN group respectively (P＜ 0.05).Further DN+CDC rats had decreased both p21 and p27 protein expressions in glomeruli,but DN+ ARB rats only had decreased p27 protein expression (all P ＜ 0.05).Conclusions 12-LO may induce both p21 and p27 protein expression in DN glomeruli,but Ang Ⅱ may induce only p27 expression.

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.
Amidohydrolases ; isolation & purification ; metabolism ; Arthrobacter ; enzymology ; Bacterial Proteins ; isolation & purification ; metabolism ; Chromatography, DEAE-Cellulose ; methods

OBJECTIVETo study the role of O(6)-methylguanine-DNA methyltransferase (hMGMT) in the development of human lung cancer.

METHODSReverse transcription-polymerase chain reaction (RT-PCR) method was applied to measure hMGMT mRNA expression in 150 lung cancer specimens, 40 normal lung tissues, and in the peripheral mononuclear blood cells from 50 lung cancer cases and 50 normal controls. The protein expressions of p53, C-MYC and K-RAS were assessed by immuno-histochemistry. The effects of some exposure factors on the expression of hMGMT gene were analyzed. The relationships between hMGMT gene and cancer related genes p53, C-MYC and K-RAS were investigated.

RESULTSThe mRNA of hMGMT was low or absent in 49 of 150 (32.7%) lung cancer specimens, whereas 2 of 40 (5%) normal lung tissues had reduced the levels of hMGMT mRNA. The low expression of hMGMT seemed to be a risk factor of lung cancer, with a OR of 9.22 (2.05-57.65). Reduced expression levels of hMGMT mRNA were observed in 10 of 50 (20%) lung cancer patients' peripheral mononuclear blood cells, and 2 of 50 (4%) blood cells among normal controls. When investigating the exposure factors which affecting the expression of hMGMT gene, we noticed that smoking was suppressing the expression of hMGMT gene. Interestingly, over-expression of K-RAS oncogene was significantly correlated with low expression of hMGMT (P < 0.05). However, the expressions of p53 and C-myc were not correlated with the status of hMGMT gene.

CONCLUSIONhMGMT might play an important role in the development of human lung cancer. Low expression of hMGMT gene seemed to be a risk factor for lung cancer which could be used as a valuable biomarker on susceptibility of human lung cancers.

Adult ; Aged ; Biomarkers, Tumor ; Carcinoma, Squamous Cell ; enzymology ; genetics ; China ; epidemiology ; DNA Repair ; genetics ; Female ; Genes, ras ; genetics ; Humans ; Lung Neoplasms ; enzymology ; genetics ; Male ; Middle Aged ; O(6)-Methylguanine-DNA Methyltransferase ; biosynthesis ; genetics ; metabolism ; Point Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Smoking ; adverse effects ; ras Proteins ; biosynthesis ; genetics

This study was purposed to investigate the angiogenin (ANG) expression in COS-7 cells and its biological activity. The gene of angiogenin was obtained from mononuclear cells of peripheral blood by using RT-PCR and inserted into eukaryotic expression vector of pcDNA3.1. After being transfected into COS-7 cells, the recombinant ANG was identified by Western blot assay. The function of promoting proliferation of ANG to ECV304 cells was detected by MTT method, and its activity of vascularization was analyzed by chick embryo chorioallantois treated by the culture supernatant after transfection with pcDNA3.1-ang. The result showed that recombinant ANG was expressed in COS-7 cells after transfection for 24 to 36 hours. It could specifically react with monoclonal antibody against ANG. The recombinant ANG could obviously promote the proliferation of ECV304 cells. In contrast with the control group, the culture supernatant of pcDNA3.1-ang transfected group could stimulate the angiogenesis in embryo chorioallantois. It is concluded that the ang transiently expresses in COS-7 cells, and its expression product obviously stimulates the cell proliferation and angiogenesis.
Angiogenesis Inducing Agents ; pharmacology ; Animals ; COS Cells ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Endothelial Cells ; cytology ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Ribonuclease, Pancreatic ; biosynthesis ; genetics ; pharmacology ; Transfection

OBJECTIVETo investigate whether a simulated He-O2 saturation dive to 65 msw would affect oxidative balance in humans.

METHODSSeven divers participated in a simulated saturation dive to 0.75 MPa (65 msw). 24-h urine samples were collected twice before, twice during, and twice after the dive, then were analyzed for contents of superoxide dismutase (SOD), malondialdehyde (MDA), total amino acid (T-AA) and total anti-oxidant capacity (T-AOC). Meanwhile, total urine volume and body weight were measured.

RESULTSThe content of T-AA was higher. (P < 0.05) than the base value in final decompression, but reverse to normal at one week after decompression. There were no changes in contents of SOD, MDA and T-AOC during and after the dive compared with their basic value. Total urine volume was lower (P < 0.05, vs basic value) at first day in chamber, then returned to normal. Body weight gradually increased after compression till the end of decompression (higher than basic value, P < 0.05).

CONCLUSIONThese data indicate that simulated saturation dive to 65 msw may not induce obvious oxidative damage, but it is necessary to monitor 24-h urine volume and oxidative sress by time in order to prevent from tissue injury.

Adult ; Amino Acids ; urine ; Decompression ; Diving ; physiology ; Helium ; chemistry ; Humans ; Male ; Malondialdehyde ; urine ; Oxidative Stress ; physiology ; Oxygen ; adverse effects ; chemistry

Objective To observe the effects of Qijingmingmu decoction granule on the expression of mitogen-activated protein linase (MAPK) signal pathway in fibroblasts of conjunctivochalasis (CCH) under the stimulation of tumor necrosis factor (TNF-α) and to explore the pathogenesis and effective treatment method of CCH.Methods CCH conjunctival fibroblasts were cultured in vitro and divided into CCH group,CCH + TNF-α group and CCH + TNF-α + Qijingmingmu decoction group.CCK-8 assay was used to determine the effective concentration of Qijingmingmu decoction granule,and 10-2 mg · L-1 TNF-α was added in the cultured fibroblasts of the latter two groups,followed by interference with Qijingmingmu decoction granule for 48h.The expression of MAPK signal pathway related protein and mRNA were detected by ELISA,Western Blot and RT-PCR.Then the results were statistically analyzed.Results CCK-8 assay showed the effective concentration of Qijingmingmu decoction granule was 1.61g · L-1.And there were significant differences in the total A values (A450) of extracellular regulated protein kinase (ERK),c-jun N-terminal kinase (JNK),p38 mitogen activated protein kinase (p38 MAPK) and their phosphorylation levels among the three groups (all P ＜ 0.05).Moreover,TNF-α could significantly up-regulate the expression of ERK1/2,JNK1/2,p-JNK1/2,p38 MAPK and p-p38 MAPK in CCH fibroblasts (all P ＜0.05),while Qijingmingmu decoction down-regulated their expressions in CCK fibroblasts after TNF-α stimulation (all P ＜ 0.05).Furthermore,the total differences in p-ERK1/2,p38 MAPK and p-p38 MAPK protein were significant in the three groups (all P＜0.05);and TNF-α could significantly up-regulate the expression of p-ERK1/2,p-JNK1/2,p38 MAPK and p-p38 MAPK protein in CCH fibroblasts (all P ＜0.05),while Qijingmingmu decoction down-regulated p-ERK1/2 and p38 MAPK expressions in CCK fibroblasts after TNF-α stimulation (both P ＜ 0.05).In addition,the total differences in ERK1/2 and p38 MAPK mRNA were significant in the three groups (both P ＜ 0.05);and TNF-α could significantly up-regulate the expression of ERK1/2 and p38 MAPK mRNA in CCH fibroblasts (both P ＜ 0.05),while Qijingmingmu decoction down-regulated p38 MAPK mRNA expressions in CCK fibroblasts after TNF-α stimulation (P ＜ 0.05).Conclusion The inflammatory factor TNF-α can up-regulate the expressions of MAPK signal pathway related protein and mRNA in CCH fibroblasts,resulting in the occurrence and development of CCH,and meanwhile Qijingmingmu decoction granule can downregulate the expression of MAPK signal pathway to play the therapeutic role in CCH to some extent.

Objectives To evaluate the role of PCSK9 (proprotein convertase subtilisin kexin type 9) on the lipid accumulation and kidney injury of C57BL/6 mice. Methods The 24 h urine of 12 weeks old wide type C57BL/6 mice and PCSK9 knockout (KO) mice were collected through a metabolic cage, followed by perfusion and sacrifice. Urinary microalbumin?to?creatinine ratio (UACr), total cholesterol and triglyceride in kidney tissues were measured by ELISA. BODIPY 493/503 staining and standard transmission electron microscopy (TEM) of kidney tissues was performed for evaluating lipid accumulation and podocyte foot effacement in the kidney. Kidney tissues were also evaluated by PAS stain and TUNNEL stain. PCSK9, podocin and nephrin were quantified through real?time PCR, and the Bcl?2, Bax and cleaved caspase 3 were evaluated by Western blotting. Results Total cholesterol and triglyceride contents were higher in the kidneys of PCSK9 KO mice than controls (P<0.05). The level of lipid accumulation in glomeruli and tubules through BODIPY 493/503 stain, and the amount of lipid drop in TEM were more serious in PCSK9 KO mice. UACr and podocyte foot process effacement were increased, and the transcription of podocin and nephrin were decreased in the kidneys of PCSK9 KO mice (all P<0.05). The expression of Bcl?2 was decreased, and Bax and cleavedcaspase 3 were increased in the kidney samples of PCSK9 KO mice. Conclusion PCSK9 might be reversely involved in lipid homeostasis and accumulation, resulting in injury and apoptosis in the kidneys of C57BL/6 mice.

BACKGROUND: Acute liver failure (ALF) caused by viral and non-viral hepatitis is often accompanied with severe metabolic disorders, the accumulation of toxic substances and continuous release and accumulation of a large number of endogenous toxins and inflammatory mediators. The present study aimed to investigate the effects of various combined non-biological artificial liver treatments for patients with acute liver failure (ALF) complicated by multiple organ dysfunction syndrome (MODS). METHODS: Thirty-one patients with mid- or late-stage liver failure complicated by MODS (score 4) were randomly divided into three treatment groups: plasmapheresis (PE) combined with hemoperfusion (HP) and continuous venovenous hemodiafiltration (CVVHDF), PE+CVVHDF, and HP+CVVHDF, respectively. Heart rate (HR) before and after treatment, mean arterial pressure (MAP), respiratory index (PaO2/FiO2), hepatic function, platelet count, and blood coagulation were determined. RESULTS: Signifi cant improvement was observed in HR, MAP, PaO2/FiO2, total bilirubin (TBIL) and alanine aminotransferase (ALT) levels after treatment (P<0.05). TBIL and ALT decreased more signifi cantly after treatment in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.01). Prothrombin time (PT) and albumin were signifi cantly improved only in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.05). TBIL decreased more significantly in the PE+HP+CVVHDF group than in the HP+CVVHDF and PE+CVVHDF groups (P<0.05). The survival rate of the patients was 58.1％ (18/31), viral survival rate 36.4％ (4/11), and non-viral survival rate 70％ (14/20). CONCLUSION: Liver function was relatively improved after treatment, but PE+HP+CVVHDF was more efficient for the removal of toxic metabolites, especially bilirubin. The survival rate was significantly higher in the patients with non-viral liver failure than in those with viral liver failure.