Effects of pargyline on histone methylation in promoter and enhancer regions and transcription of CYP3A4/3A7 / 药学学报

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-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.

Effect of FK506 nanospheres on regeneration of allogeneic nerve after transplant

OBJECTIVE@#To discuss effect of FK506 nanospheres used at different time on the regeneration of allogeneic nerve after transplant.@*METHODS@#Single emulsion-solvent evaporation method (O/W) was adopted to prepare the FK506 nanospheres and the tibial nerve of rats after allogeneic transplantation. FK506 nanospheres were used in group A after operation immediately, in group B in 24 h after operation, and in group C in 3 d after operation while FK506 nanospheres were not used in group D; in the 4th, 8th and 12th week after operation respectively, general observation of transplanted nerves, histological examination, image analysis of myelinated fibers, wet-weight determination of musculi triceps surae, retrogradely labeling of neurons by the fluorescein and electrophysiological comparison of bilateral tibial nerve were carried out.@*RESULTS@#FK506 nanospheres can be degraded and absorbed quickly. The neural regenerations in group A and B were similar, which were both much better than those in group C and D. The difference was statistically significant and so was the difference between group C and D.@*CONCLUSIONS@#Drug release rate of FK506 nanospheres is accordant with the regeneration law of damaged nerves and the local application can promote the regenerations of nerves. The effect would be better if the drug is used in earlier period (within 24 h).

Over-expression of testis-specific expressed gene 1 attenuates the proliferation and induces apoptosis of GC-1spg cells / 华中科技大学学报（医学）（英德文版）

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.

Over-expression of testis-specific expressed gene 1 attenuates the proliferation and induces apoptosis of GC-1spg cells / 华中科技大学学报（医学）（英德文版）

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.

High frequency of regulatory T cells among HIV type 1-infected men who have sex with men correlates with disease progression / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Regulatory 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.</p><p><b>METHODS</b>Fifty-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Increased proportions and decreased numbers of CD4(+) Tregs are associated with HIV progression, and their functions may impair with the progression of HIV infection.</p>

Hyperammonemia-induced hepatic injury in rats: characterization of a new animal model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish an accurate new rat model of hyperammonemia-induced liver injury for use in studies of the molecular mechanisms underlying acute liver failure (ALF).</p><p><b>METHODS</b>Twenty-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).</p><p><b>RESULTS</b>ALF 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.</p><p><b>CONCLUSION</b>ALF 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.</p>

Effects of cefodizime on chemokines of liver tissues in mice with immunological hepatic injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chronic 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>Cefodizime 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.</p>

Construction of a eukaryotic expression vector expressing human IFN-gamma and its inhibitory effect on HBV replication in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Compared 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.</p><p><b>CONCLUSION</b>We have successfully constructed the eukaryotic expression vector expressing human IFN-gamma, which provides a basis for anti-HBV gene therapy using human IFN-gamma.</p>

Coxsackievirus B3 infection induced viral myocarditis by regulating the expression pattern of chemokines in cardiac myocytes

Viral myocarditis is a common cardiovascular disease, which has greatly threatened human health. However, up to now, the pathogenesis of viral myocarditis has been unclear, which leads to the lack of its effective treatments. To investigate the role of chemokines in pathogenesis of viral myocarditis, mRNA expression for a panel of 19 chemokines detected by RT-PCR in myocardial tissue of BALB/c mice that were inoculated intraperitoneally with coxsackievirus B3. Moreover primary cultured cardiac myocytes were infected with coxsackievirus B3 following extraction of RNA, from myocytes the expression of 19 chemokines was detected by by RT-PCR. Our results showed that there was much difference in the expression pattern of chemokines in myocardial tissue between infected mice with viral myocarditis and uninfected control mice. The expression of chemokines was varied significantly in clusters in myocardium post coxsackievirus B3 infection. There were also complexity and imbalance in the change of the expression of chemokines. In the meantime, Coxsackievirus B3 infection also influenced the expression pattern of chemokines in cardiac myocytes in vitro. However the expression of monocyte chemoattractant protein-1 alone was upregulated in cardiac myocytes post coxsackievirus B3 infection in the 19 detected chemokines. The chemokine expression pattern changed in complexity and imbalance manner both in myocardium and in primary cultured cardiac myocytes after coxsackievirus B3 infection. Coxsackievirus B3 infection may start viral myocarditis by regulating the expression pattern of chemokines in cardiac myocytes. MCP-1 may be one of key chemokines in the initial stage of viral myocarditis

Complete human immunodeficiency virus-1 specific T lymphocyte response to Chinese human immunodeficiency virus-1 B/C chronic infectors / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Twenty-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.</p>

Effect of age on ED50 of rocuronium for intratracheal intubation in female patients / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Forty 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

Analysis on the decline of hepatitis C virus RNA in the blood using a mathematical model / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a mathematical model of hepatitis C virus (HCV) replication and develop a working theory for antiviral therapy in order to understand the dynamics of HCV replication.</p><p><b>METHODS</b>Peripheral blood cells of 4 hepatitis C patients were cultured. Quantities of the HCV were detected every 15 min by real-time PCR. The data were analyzed using SPSS software. A mathematical functional relationship between HCV RNA and the time lapse was established.</p><p><b>RESULTS</b>The quantity of HCV RNA declined and it fell into a mathematical model: Y=3E+0.8e(-0.5467x) (r=0.9547). The estimated virion half-life was 45 min on the average.</p><p><b>CONCLUSIONS</b>The decline of HCV RNA in the blood is not of a linear trend and the HCV RNA lasts a longer time although the speed of the decline is faster than that in vivo.</p>

The comparison of the effects of three kinds of anti-retroviral therapy on patients with human immunodeficiency virus infection in acquired immune deficiency syndrome area / 中华传染病杂志

0.05);however there were significant difference between D4T+DDI+NVP group and AZT+DDI+NVP group(P

Influence of inserting glycines on biological properties of HIV Tat-(Gly)n-thymidine kinase fusion proteins / 第二军医大学学报

Objective: To study the influence of inserting glycines(Gly) on biological properties of HIV Tat-(Gly)n-thymidine kinase (-TK) fusion proteins. Methods: Different fragments containing 0, 2, 4 or 6 Gly were inserted between the HIV Tat gene and TK using gene splicing by overlap extension (SOEing) PCR, and the products were cloned into PBK vector. The vectors were then transferred into E. coli after sequencing. After IPTG induction, bacilli were collected and destructed by ultrasound; the fusion protein was collected and identified by monoclonal antibody of HIV protein. HepG2 cells were incubated with DMEM supplemented with 1 μg/ml fusion protein containing 0, 2, 4 or 6 Gly for 24 h. HepG2 cells of different groups were detected by immunofluoreseence assay with HIV Tat monoclonal antibody; the apoptosis rate of HepG2 cells was determined by cell flow cytometry after they were incubated with gencilovir (10 μg/ml) for 3 d and the survival rate of cells was recorded by trypan blue in different groups. Results: The recombined genes containing 0, 2, 4 or 6 Gly were successfully constructed, inserted into PBK vectors, and expressed into E. coli. Their proteins were obtained and purified. The level of fluorescence in different groups was similiar, but the cell survival rate and apoptosis rate were different. The highest apoptosis rate was 14.77%, which was found in the group containing 4 Gly, followed by 12.69% in 2 Gly group, 8.31% in HIV Tat-TK group, 4.36% in 6 Gly group, and 1.0% in group containing no Gly. Significant differences were found between each 2 groups (P<0.05). Trypan blue showed similar results in the cell death rate of different groups: the highest cell death rate was 80.2%, which was found in the group containing 4 Gly, followed by 65.4% in 2 Gly group, 58.4% in HIV Tat-TK group, 56.7% in 6 Gly group, and 9.1% in the group containing no Gly. Conclusion: The number of Gly inserted into HIV Tat-TK protein does not alter the transcellular function of upstream Tat protein, but does substantially influence the TK protein-mediated cytoxic effects of gencilovir, and the influence is the smallest when 4 Gly are inserted.

Construction and expression of the recombinant human immunodeficiency virus Tat gene and analysis on its biological characteristics / 中华传染病杂志

Objective To determine the influence of protein fusion on the biological characteris- tics of hymidine kinase(TK)and human immunodeficiency virus(HIV)Tat recombinant protein. Methods By utilizing polymerase chain reaction(PCR)technique,different fragments containing two,four or six glycines(Gly)were inserted between the HIV Tat gene and TK,and cloned into PBK vector.After testified by sequencing,the vectors were transfected into E coli.After induced by iso- propyl thiogalactose(IPTG),bacilli were collected and destructed by ultrasonic,the fusion proteins were determined by monoclonal antibody against HIV protein.HepG2 cells were incubated in DMEM supplement with 10?g/mL HIV-Gly(n)-TK(n=0,2,4,6)fusion protein,TK-HIV Tat and only HIV Tat.HepG2 cells in different groups were detected by immunofluorescence assay 24 hours after transduction with HIV Tat monoclonal antibody.The rate of apoptosis after cells were incubated with gencilovir(10?g/mL)for 3 days was determined by cell flow cytometry,while survival cell ratio was recorded by trypan blue.The data were analyzed by statistics(t-test).Results The Tat-Gly(n)-TK (n= 0,2,4,6)recombinant genes were constructed and inserted into PBK vectors,which were expressed in E coli and then purified.Cells in different groups,which were incubated with Tat-Gly (n)-TK(n=0,2,4,6)fusion proteins,Tat-TK fusion protein,TK-Tat fusion proteins or only Tat proteins respectively,were detected by immunofluorescence assay.The intensities of fluorescence in different groups were almost same,but the ratios of cell survival or apoptosis were different.The highest ratio of cells apoptosis(14.77%)was in the group that cellular culture medium was mixed with Tat-Gly(4)-TK fusion protein,followed by the groups containing 6,2 glycines or no TK gene in genes(4.30%,12.69% and 1.03%,respectively).There were significant differences between each 2 groups among the all groups(t-test,P