Biliary atresia is a progressive obliterative cholangiopathy, but the etiology of this disorder remains uncertain. Identifying genes specifically expressed in biliary atresia and analyzing the pattern of expression may lead to a better understanding of the pathogenesis. Liver tissues were taken from a recipient with biliary atresia and a normal donor during liver transplantation. Total RNA was extracted from each sample and reversely transcribed to cDNA. Then radiolabeled cDNA probe pools were made by random primed DNA labeling method and used for screening of differentially expressed genes by hybridizing with expressed sequence tags (EST) dot blot panel. Northern blot hybridization was done to confirm that these genes are also differentially expressed in other liver tissues. Among 1,730 EST clones, 26 cDNA clones were significantly overexpressed in biliary cirrhosis, while 2 clones were significantly decreased in biliary atresia. By Northern blot hybridization, the results of tissue inhibitor of metalloproteinase (TIMP)-1 and IGFBP-2 were well correlated with differential EST screening (DES). This study identified the pattern of differentially expressed genes in the biliary cirrhosis due to biliary atresia using DES technique.
Biliary Atresia/*genetics ; Blotting, Northern ; Gene Expression Profiling/*methods ; Gene Library ; Human ; Insulin-Like Growth Factor-Binding Protein 2/genetics ; Tissue-Inhibitor of Metalloproteinase-1/genetics

Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; Genes, Retinoblastoma ; genetics ; Genes, erbB-1 ; genetics ; Genes, p53 ; genetics ; Glioma ; genetics ; pathology ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Tumor Suppressor Proteins ; genetics

OBJECTIVETo investigate the expressions of inflammation- and fibrosis-related genes in perinephric and subcutaneous adipose tissues in patients with adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome.

METHODSThe perinephric and subcutaneous adipose tissues adipose tissues were obtained from 8 patients with ACTH-independent Cushing's syndrome undergoing laparoscopic retroperitoneal adrenalectomy. Real-time PCR was used to detect the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metallopeptidase 2 (MMP-2), TIMP metallopeptidase inhibitor 1 (TIMP-1), early growth response 1 (EGR1), CCAAT/enhancer binding protein β(CEBPβ), uncoupling protein 1(UCP-1), PPARγ coactivator 1 alpha (PGC1α) and cell death-inducing DFFA-like effector a (CIDEA).

RESULTSThe mRNA level of CIDEA was significantly higher in the perinephric adipose tissue (peri-N) than in the subcutaneous adipose tissue (subQ) (P<0.05). The expressions of CEBPβ, UCP-1, and PGC1α mRNA in the peri-N were similar with those in the subQ. The expressions of IL-6, TIMP1 and EGR1 mRNA in the subQ were significantly higher than those in the peri-N (P<0.05). No significant difference in TNF-α and MMP-2 mRNA levels was found between peri-N and subQ.

CONCLUSIONThe expression levels of the inflammation- and fibrosis-related genes are higher in the subQ than in the peri-N of patients with ACTH-independent Cushing's syndrome, suggesting that chronic exposure to endogenous hypercortisolism may cause adipose tissue dysfunction.

Adrenalectomy ; Adrenocorticotropic Hormone ; CCAAT-Enhancer-Binding Protein-beta ; metabolism ; Cushing Syndrome ; metabolism ; surgery ; Early Growth Response Protein 1 ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Real-Time Polymerase Chain Reaction ; Subcutaneous Fat ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Uncoupling Protein 1 ; metabolism

BACKGROUND: Subjects with growth hormone-deficiency (GHD) have increased cardiovascular mortality, and growth hormone (GH) replacement may modulate cardiovascular disease risk. Therefore, we evaluated the effects of GH administration on the markers of cardiovascular disease in subjects with GHD. METHODS: 37 subjects (12 men and 25 women) with GHD and 65 normal subjects were enrolled in this study. GH or placebo were given for 3 months at a dose adjusted for normal serum insulin-like growth factor-I (IGF-I) level. Height, weight, waist circumference, hip circumference, lean body mass, fat mass, blood pressure, fasting blood glucose, IGF-I, lipid profile, uric acid, C-reactive protein (CRP), plaminogen activator inhibitor-1 (PAI-1), apolipoprotein AI, and quality of life-assessment of growth hormone deficiency in adults (QoL-AGHDA) were measured at baseline and month 3. RESULTS: Subjects with GHD showed higher levels of triglyceride, CRP, and PAI-1, but lower level of fasting glucose than normal subjects. Fat mass, CRP, and PAI-1 levels decreased in GH recipients (fat mass; 21.9+/-6.6 to 21.3+/-6.7%, p<0.05, CRP; 2.73+/-2.11 to 1.47+/-1.29 mg/L, p<0.001, PAI-1; 48.9+/-33.2 to 31.6+/-28.5 ng/mL, p<0.05). Fasting blood glucose and total cholesterol levels increased in GH recipients (fasting blood glucose; 4.58+/-0.46 to 4.81+/-0.36 mmol/L, p<0.05, total cholesterol; 5.36+/-1.31 to 6.17+/-1.12 mmol/L, p<0.01). Placebo recipients showed decrease in waist-hip ratio (0.93+/-0.05 to 0.92+/-0.04, p<0.05) and increase in fasting blood glucsoe (4.63+/-0.38 to 4.89+/-0.45 mmol/L, p<0.05) and uric acid (319.6+/-89.2 to 335.6+/-89.2 micro mol/L, p<0.05). QoL-AGHDA score improved in both groups (GH recipients; 10.0+/-6.0 to 7.4+/-5.5, p<0.01, placebo recipients; 9.8+/-4.4 to 6.7+/-3.4, p<0.05). CONCLUSION: Our results demonstrated favourable effects of GH on cardiovascular disease through modulating CRP and PAI-1 plasma level in subjects with GHD.
Adult* ; Apolipoprotein A-I ; Blood Glucose ; Blood Pressure ; C-Reactive Protein ; Cardiovascular Diseases* ; Cholesterol ; Fasting ; Glucose ; Growth Hormone* ; Hip ; Humans ; Insulin-Like Growth Factor I ; Male ; Mortality ; Plasma ; Plasminogen Activator Inhibitor 1 ; Triglycerides ; Uric Acid ; Waist Circumference ; Waist-Hip Ratio

OBJECTIVE: To investigate the ING1 gene mutation status in human laryngeal squamous cell carcinoma(LSCC), and the association of p33(ING1b) protein expression with p53 protein expression. METHOD: DNA of LSCC tissue was extracted, and nucleotide of the second exon was amplified and sequenced to determine the chromosome status. The p23(ING1b) and p53 protein expression were detected by immunohistochemistry and the association between them were analyzed. RESULT: No mutation was detected in ING1 gene, but a single polymorphism from GGG to AGG at codon 170 of ING1 gene was found in 2 of the 25 LSCC tissues. The immunohistochemical analysis showed that 4 had positive p33(ING1b) expression. No association was found between p33(ING1b) expression and LSCC clinical features, or between p53 and clinical features. However, significant difference was found between p33(ING1b) and p53 expression. p33(ING1b) tended to be negative in p53 expression positive tissue. CONCLUSION ING1 gene mutation appears rare in LSCC. In normal physical condition, p33(ING1b) may play a synergistic effect with p53 protein.
Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Female ; Genes, Regulator ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Tumor Suppressor Protein p53 ; metabolism ; Tumor Suppressor Proteins ; genetics

OBJECTIVESTo investigate the effect of rhein on endothelial plasminogen activator inhibitor-1 (PAI-1) mRNA expression and protein production induced by transforming growth factor beta1 (TGFbeta1), and to explore the mechanism of the protective action of rhein on endothelial cells.

METHODSA human umbilical endothelium derived cell line (ECV-304) from ATCC was used in this study. The PAI-1 mRNA expression and protein synthesis in the endothelial cells were detected by Northern blot and flow cytometry analysis, respectively. The activity of phospho-p44/p42 MAP kinase induced by TGFbeta1 was determined by immunoprecipitation analysis and western blot.

RESULTSTGFbeta1 rapidly increased PAI-1 mRNA expression in the endothelial cells, and this effect lasted at least 24 hours. The upregulation of PAI-1 mRNA expression induced by TGFbeta1 in endothelial cells was inhibited by rhein in a dose-dependent manner. In addition, rhein inhibited endothelial PAI-1 protein production. Further study revealed that rhein had a significant inhibitory effect on the activity of phospho-p44/p42 MAP kinase induced by TGFbeta1 in human endothelial cells.

CONCLUSIONSOur results showed that rhein may have a protective effect on the endothelial dysfunction by inhibiting overexpression of PAI-1, indicating a way for the treatment of vascular diseases.

Anthraquinones ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; drug effects ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; metabolism ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; analysis ; Transforming Growth Factor beta ; antagonists & inhibitors ; Transforming Growth Factor beta1

BACKGROUNDNeural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS), which are active during development and maintain homeostasis and tissue integrity throughout life. Microglias are an immune cell population resident in the CNS, which have crucial physiological functions in the developing and adult CNS. This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.

METHODSMicroglia and NSCs were co-cultured in 24-well insert plates. NSCs were plated in the bottom of the well and microglia in the insert. Fluorescent staining, Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.

RESULTSCo-culture of microglia and NSCs promoted astrogliogenesis from NSCs. Several key genes, such as Notch 1, Notch 2, Notch 3, Hes 5, and NRSF were downregulated, while the critical genes Id1 and Id2 were upregulated. BMP2 and FGF2 were upregulated.

CONCLUSIONMicroglias act as a regulator of NSCs astrogliogenesis.

Animals ; Astrocytes ; cytology ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Coculture Techniques ; methods ; Fibroblast Growth Factor 2 ; genetics ; Inhibitor of Differentiation Protein 1 ; genetics ; Inhibitor of Differentiation Protein 2 ; genetics ; Microglia ; cytology ; metabolism ; Microscopy, Fluorescence ; Neural Stem Cells ; cytology ; metabolism ; Rats ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study effects of alternative transcripts of ING1 transfection on human cancer cell lines.

METHODSp47/ING1A and p33/ING1B expression vehicles were constructed and introduced into a human breast cancer cell line MCF-7 and a human lung cancer cell line PAa, both expressing wild-type p53 protein. Growth characteristics of the transfectants and potentially related genes were analyzed.

RESULTSThe levels of p47/ING1A and p33/ING1B protein elevated respectively in tumor cells of MCF-7 and PAa after transfected with p47/ING1A and p33/ING1B, and the latter was much higher than that of the former. Ectopic overexpression of p33/ING1B effectively blocked tumor cell growth and arrested cells in the G(0) approximately G(1) phase of the cell cycle (P < 0.01), while p47/ING1A gave no effect on cell growth or cell cycle. Tumor cells overexpressing p33/ING1B contained more p21(WAF1) protein than that of the control cells, with undisturbed p53 protein level.

CONCLUSIONSExpression of two different transcripts of ING1 may have different effects on tumor cell growth. p33/ING1B may cooperate with p53 in stimulating expression of p21(WAF1) gene, thus to arrest cell cycle and to inhibit tumor cell growth. p33/ING1B may be considered to be a candidate as a partner of p53 in gene therapy.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Alternative Splicing ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Cycle Proteins ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; DNA-Binding Proteins ; Genes, Tumor Suppressor ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Nuclear Proteins ; Protein Biosynthesis ; Proteins ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; Tumor Suppressor Proteins

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics ; Animals ; Chondrocytes/drug effects/*metabolism ; Collagen Type II/genetics/metabolism ; Fibroblast Growth Factor 2/*genetics ; Gene Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Humans ; Insulin-Like Growth Factor I/*genetics/metabolism ; Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism ; Interleukin-1/genetics/metabolism ; Matrix Metalloproteinase 13/genetics/metabolism ; Matrix Metalloproteinase 3/genetics/metabolism ; Osteoarthritis/*therapy ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Transfection

Thickening of tubular basement membrane and progressive tubulointerstitial fibrosis has been reported as important components of diabetic nephropathy, In order to investigate the mechanisms of tubulinterstitial changes in diabetic nephropathy, we evaluated the effects of a high concentration of glucose(25mM; 450mg/dL) on glucose transporter GLUT1 level, fibronectin production and tissue inhibitors of metalloproteinases (TIMP)-1 concentration in renal tubular(LLC-PK1) cells. As the effect of high glucose-induced alteration in LLC-PK1 cells, the expression of facilitative glueose transporter, GLUT1 was decreased after longer than 24-hours exposure to 25mM glucose, compared to control(5.6mM). The administration of protein kinase C (PKC) inhibitor GF109203X(10 microM) did not show significant effect on high glucose-induced decrease of GLUT1 level. On western blot analysis of fibronectin production, The exposure of LLC-PK cells to 25mM glucose for 48 hours significantly increasc4 fibro- nectin production, dose-dependently. The addition of GF102903X at the concentration of 10pM induced the significant increase of fibronectin level in LLC-PK1cells under glucose-free condition, whereas there was no significant effect on the high glucose-induced increase of fibronectin production. The addition of anti-TGF-beta antibody at 30 microgram/mL partly inhibited the high glucose-induced increase of fibronectin production. Concerning the changes of tissue inhibitor of metallo-proteinase(TIMP)-1 levels in the presence of high glucose, the exposure to high glucose for 24 and 43 hours increased TIMP-1 levels in culture supernatant of LLC-PK1 cells, dose-dependently. The TIMP-1 levels of 48-hour exposure to 15 and 25mM glucose were also significantly higher than those of 24-hourexposure. The treatment with 10 microM GF102903X or 30 microgram/mL anti-TGF-Beta antibody had no significant effects on TIMP-1 levels measured under the high glucose culture condition. In conclusion, the expression of facilitative glucose transporter, GLUT1 is inhibited and the production of fibronectin is increased in renal tubular cells cultured in the presence of high concentration of glucose, which is partly mediated by TGF-beta. The TIMP-1 level is also increased under high glucose culture condition. The enhanced productions of fibronectin and TIMP-1 of renal tubular cells under high glucose concentration may contribute to tubulointerstitial fibrosis that occurs in diabetic nephropathy.
Animals ; Basement Membrane ; Blotting, Western ; Diabetic Nephropathies ; Epithelial Cells* ; Extracellular Matrix* ; Fibronectins ; Fibrosis ; Glucose Transport Proteins, Facilitative ; Glucose* ; LLC-PK1 Cells ; Metalloproteases ; Protein Kinase C ; Swine ; Tissue Inhibitor of Metalloproteinase-1 ; Transforming Growth Factor beta