This study was designed to investigate effects of pargyline on histone methylation in the promoter and enhancer regions and transcription of cytochrome P450 3A4/3A7 (CYP3A4/3A7) gene. Human primary fetal liver cells were isolated, cultured and randomly divided into several groups including control, solvent, pargyline low, middle, high dose (treated with 0.6, 1.2, 2.4 mmol·L^-1). HepG2 cells were cultured and treated with 0.03, 0.3, 3 mmol·L^-1 pargyline. After 48 hours, total RNAs were prepared from the cells to determine the expression of CYP3A mRNA in primary fetal cells and HepG2 cells with real-time quantative PCR (qPCR). HepG2 cells were cultured and then treated with 3 mmol·L^-1 pargyline for 48 hours. The chromatin immunoprecipitation (ChIP) assay was performed with dimethylation of histone H3 at lysine 4 (H3K4me2), and IgG antibodies respectively. The precipitated DNA was resuspended and used for qPCR. Primers were used to detect different regions of CYP3A4/3A7 promoter and enhancer. Occupancy of H3K4me2 was shown as percent of input DNA relative to control cells. The results suggested that pargyline has an effect on primary fetal liver cells and HepG2 cells proliferation. The level of CYP3A7 was markedly enhanced in human primary fetal liver cells by treatment with 1.2, 2.4 mmol·L^-1 of pargyline (P<0.05, P <0.01) and the levels of CYP3A4/3A7 were remarkably enhanced by treatment with 3 mmol·L^-1 of pargyline in HepG2 cells (P<0.001) compared with solvent control. Occupancy of H3K4me2 on human CYP3A4 promoter (-362 to +53) and enhancer segment (-7 836 to -6 093) harbored the overlapping hepatocyte nuclear factors 4A (HNF4A) binding site compared with a negative control. Occupancy of H3K4me2 on human CYP3A7 promoter (-163 to +103) and enhancer segment (-4 054 to -3 421, -6 265 to -6 247) overlapped with glucocorticoid receptor (GR) binding site. In conclusion, the enriched H3K4me2 in the promoter and enhancer regions was induced by pargyline with HNF4A or GR binding site in CYP3A4/3A7 gene to activate the corresponding genes.

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

OBJECTIVETo establish an accurate new rat model of hyperammonemia-induced liver injury for use in studies of the molecular mechanisms underlying acute liver failure (ALF).

METHODSTwenty-six Sprague-Dawley rats were administered D-galactosamine (400 mg/kg) and endotoxin (50 mug/kg) via intraperitoneal injection to induce ALF and sacrificed at 12 h post-injection (ALF-12 group, n = 10) or 24 h post-injection (ALF-24 group, n = 16). Ten rats administered physiological saline served as the control group. In addition, 20 rats were given serial oral administrations of 10% NH4Cl solution (10 ml/kg, every 8 hrs) to establish the hyperammonemia-induced liver injury model; an additional 20 rats were prepared in parallel to serve as the ALF control group (n = 10; D-galactosamine at 800 mg/kg every 6 d for 30 days) and the physiological saline control group (n = 10). Serum samples were collected from each mouse and used to detect markers of liver function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alpha-fetal protein (AFP), and gamma-glutamyltransferase (GGT), as well as blood ammonia (BA) level and prothrombin time activity (PT-A). Affects on liver histology was assessed by hematoxylin and eosin staining of resected liver tissues, and on apoptosis by TUNEL assay and calculating the apoptotic index (AI).

RESULTSALF rats showed elevated levels of ALT (1202.51+/-282.00 U/L), AST (1560.14+/-298.98 U/L), and BA (165.9+/-23.6 mumol/L) as early as 6 hrs after model establishment; these levels peaked at 12 hrs after model establishment (ALT: 774.40+/-207.65 U/L; AST: 967.60+/-121.94 U/L; BA: 143.4+/-18.1 mumol/L; P less than 0.05). No significant variations were detected in the levels of AFP (except for the ALF-24 group) or GGT. Liver tissues of the ALF-12 and ALF-24 groups showed large or diffuse hemorrhagic necroses with sinusoidal congestion or spotty bleeding, as well as increased AI. Hyperammonemia-induced liver injury rats showed elevated levels of ALT and BA as early as 6 hrs after model establishment. Similar to the ALF rats, AFP and GGT were unaffected and AI increased. However, in contrast to the ALF rats, the liver tissues of the hyperammonemia-induced liver injury rats showed no signs of hepatocyte swelling, necrosis, or inflammatory cell invasion.

CONCLUSIONALF rats and hyperammonemia-induced liver injury rats have elevated BA and marked hepatocyte necrosis. Given that reducing the level of ammonemia can improve the animal's biochemistry indexes, it is likely that hyperammonemia plays a role in acute liver injury or ALF consequent to repeated injury. The pathogenic mechanisms of repeated injury may involve promotion of hepatocyte apoptosis in conjunction with inhibition of cellular regeneration.

Animals ; Disease Models, Animal ; Hyperammonemia ; complications ; Liver Failure, Acute ; etiology ; Male ; Rats ; Rats, Sprague-Dawley

BACKGROUNDRegulatory T cells (Tregs) may play an important role in immunopathology during HIV-1 infection. Transcription factor forkhead box P3 (FoxP3) orchestrates the development of Tregs and is a useful marker to identify this population. Using a FoxP3 phenotype to define Tregs, we investigated the level and phenotype of peripheral blood natural CD4(+)Tregs and assessed the relationship between the frequencies and absolute numbers of CD4(+) Tregs and disease progression among untreated HIV-infected men who have sex with men (HIV(+) MSM) in China.

METHODSFifty-two untreated HIV(+) MSM with CD4(+) T-cell counts of ≤ 350 cells/µl or > 350 cells/µl were compared in a cross-sectional study. Twelve age-matched HIV-uninfected MSM and nine patients receiving antiretroviral therapy for at least 1 year were also included. Expression of CD25, CD127, CD45RA, CCR7 and CTLA-4 was assessed on CD4(+) Tregs using polychromatic flow cytometry.

RESULTSThe percentage of CD4(+) Tregs was increased significantly, whereas CD4(+) Tregs expressed less CTLA-4 in HIV(+) MSM compared with controls. CD4(+) Tregs displayed predominantly an effector memory phenotype (CD45RA(-) CCR7(-)), phenotypically distinct from conventional CD4(+) T cells. Moreover, the expansive frequencies of CD4(+) Tregs coincided with lower CD4(+) T-cell counts and higher viral loads whereas the absolute numbers of CD4(+) Tregs were associated with higher CD4(+) T-cell counts and lower viral loads. The expansion of Tregs was also associated with CD8(+) T-cell activation.

CONCLUSIONIncreased proportions and decreased numbers of CD4(+) Tregs are associated with HIV progression, and their functions may impair with the progression of HIV infection.

Acquired Immunodeficiency Syndrome ; immunology ; Adult ; CD4 Lymphocyte Count ; CTLA-4 Antigen ; analysis ; Cross-Sectional Studies ; Disease Progression ; HIV-1 ; Homosexuality, Male ; Humans ; Immunologic Memory ; Lymphocyte Activation ; Male ; Middle Aged ; RNA, Viral ; blood ; T-Lymphocytes, Regulatory ; immunology

BACKGROUNDChronic hepatic inflammation is characterized by the accumulation of lymphocytes as a consequence of increased recruitment from the blood and retention within the tissue at sites of infection. CXC chemokine ligand 16 (CXCL16) mRNA has been detected in both inflamed and normal liver tissues and is strongly upregulated in the injured liver tissues in a murine model. The aim of this study was to investigate the effect of cefodizime on CXCL16 mRNA of liver tissues in mice with immunological hepatic injury.

METHODSThe murine model of immunological hepatic injury was induced by Bacillus Calmette Guerin and Lipoposaccharide. The mice with immunological hepatic injury were randomly assigned to the model group, the cefodizime group and the ceftriaxone group. The three groups were continuously given agents for seven days and CXCL16 mRNA of liver tissue was determined and contrasted with the control group treated by normal saline. Reverse transcription-polymerase chain reaction was used to assay CXCL16 mRNA levels in liver tissues.

RESULTSThe expressions of CXCL16 mRNA were significantly higher in the model group and the ceftriaxone group than in the control group and the cefodizime group (P < 0.05), indicating the mice in the model group and the ceftriaxone group were immunodeficient. There was no statistical difference in the expressions of CXCL16 mRNA between the control group and the cefodizime group. Similarly, no statistical difference in the expressions of CXCL16 mRNA between the model group and the ceftriaxone group was detected (P > 0.05).

CONCLUSIONCefodizime effectively reduces the infiltration of lymphocytes into liver tissues and alleviates the liver damage by decreasing CXCL16 mRNA in liver tissues in mice with immunological hepatic injury.

Animals ; Cefotaxime ; analogs & derivatives ; therapeutic use ; Chemokine CXCL16 ; Chemokine CXCL6 ; genetics ; Chemokines ; Lipopolysaccharides ; toxicity ; Liver ; drug effects ; metabolism ; microbiology ; Mice ; Mycobacterium bovis ; physiology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the inhibitory effect of a eukaryotic expression vector expressing human IFN-gamma (pcDNA3.1- IFN-gamma) on HBV replication in hepG2.2.15 cells.

METHODSThe eukaryotic expression vector expressing human IFN-gamma was constructed using PCR and gene recombination technique. hepG2.2.15 cells were transfected with pcDNA3.1-IFN-gamma and the culture supernatant was collected to determine the expression of IFN-gamma protein by ELISA. The HBV DNA copies and the concentration of HBeAg and HBsAg were measured by fluorescence real-time PCR and ELISA kit, respectively.

RESULTSCompared with that of negative control and blank 2.2.15 cells, the concentration of HBeAg in the supernatant of 2.2.15 cells transfected with pcDNA3.1- IFN-gamma were decreased by 49%, and HBsAg concentration was lowered by 35% and 33%, respectively. A significant decrease of HBV DNA copies was observed in pcDNA3.1- IFN-gamma-transfected cells in comparison with the two control cells. No significant differences were noted in all the results between the two control groups.

CONCLUSIONWe have successfully constructed the eukaryotic expression vector expressing human IFN-gamma, which provides a basis for anti-HBV gene therapy using human IFN-gamma.

Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Interferon-gamma ; genetics ; pharmacology ; Transfection ; Virus Replication ; drug effects

Objective To study the distribution of CYP3A4 activity in 260 middle-aged gynaecologic surgical patients. Methods Midazolam (MDZ) is used as a probe of eytoehrome P450 3A4 (CYP3A4) activity by a single-point blood measurement. CYP3 A4 activity was measured by plasma ratio of 1'-hydroxymidazolam and midazolam using LC/ MS. Result The mean value of CYP3A4 activity in 260 middle-aged gynaecologic patients (aged 30-50 years) measured was 0. 46 ±0. 14 and 95% confidence interval for mean was 0. 43-0. 48. The range for normal value is 0. 18-0. 73. Conclution CYP3A4 activity is mormal distribution observed in gynaecologic patients. CYP3A4 activity variation has a little effect on the metabolism of commonly used drugs after surgery that are substrates for this enzyme.

OBJECTIVETo characterize the human immunodeficiency virus (HIV) -specific T lymphocyte responses and identify the immunodominant regions in Chinese HIV-1 recombinant subtype B/C chronic infectors at complete genome level.

METHODSTwenty-five HIV-1B/C recombinant chronic infectors were screened for their specific T lymphocyte responses to a panel of peptides corresponding to the complete HIV-1 subtype B genome by gamma interferon ELISPOT assay. Kruskal-Wallis nonparametric analysis of variance was used to test significant differences across gene regions, and Tukey pairwise analysis was used to identify differences between gene regions. Spearman rank correlation was used to assess the relation between responses. Results The order of recognized frequencies of specific T lymphocyte responses to HIV proteins was Nef>Vpr>Gag>Pol>Vpu>Env>Rev>Vif>Tat. When adjusted for protein length, Nef, Vpr, Gag, and Pol were the most intensely targeted proteins and the central region of Nef, Gag p24, Pol RT, and Vpr was most frequently recognized. No significant correlation was observed between the magnitude of IFN-gamma production of HIV-l-specific T lymphocyte responses and plasma viremia, breadth of response and CD4 counts. Conclusion The central region of Nef, Gag p24, Pol RT, and Vpr is most frequently targeted in HIV-1 B/C recombinants chronic infectors. HIV-l-specific T lymphocyte responses and plasma viremia or CD4 counts play no protective role at complete genome level in these infectors.

Adolescent ; Adult ; Asian Continental Ancestry Group ; CD4 Lymphocyte Count ; Chronic Disease ; Female ; HIV Infections ; immunology ; HIV-1 ; immunology ; Human Immunodeficiency Virus Proteins ; Humans ; Male ; T-Lymphocytes ; physiology ; Viral Load ; Young Adult

OBJECTIVETo determine the half-effective dose (IED50) of rocuronium for intratracheal intubation in female patients of different ages by sequential experiments and evaluate the effect of age on IED50 of rocuronium.

METHODSForty ASA class I-II female patients undergoing elective surgery under general anesthesia were randomly divided (n = 20) into young patient group and elderly patient group. The intratracheal intubation dose was divided into 4 grades by geometric progression, namely 0.24, 0.29, 0.35, and 0.42 mg/kg in the young patient group and 0.22, 0.26, 0.31, and 0.37 mg/kg in the elderly group. The IED(50) and 95% confidence interval (95%CI) of rocuronium during intubation in both groups were determined by sequential experiments.

RESULTSThe IED50 was 0.284 mg/kg in the elderly patient group, which was 91% that of in the young patient group (0.312 mg/kg), showing significant difference between the two groups (P < 0.05).

CONCLUSIONThe IED50 of rocuronium is significantly lower in elderly female patients than in young female patients, suggesting the necessity of reducing the dose of rocuronium accordingly in anesthesia induction in elderly female patients.

Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Androstanols ; administration & dosage ; Dose-Response Relationship, Drug ; Female ; Humans ; Intubation, Intratracheal ; Middle Aged ; Neuromuscular Nondepolarizing Agents ; administration & dosage ; Sex Factors