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.

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 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

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

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.

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

OBJECTIVETo 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.

METHODSPeripheral 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.

RESULTSThe 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.

CONCLUSIONSThe 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.

Adult ; Half-Life ; Hepacivirus ; Hepatitis C, Chronic ; blood ; virology ; Humans ; Models, Theoretical ; Nonlinear Dynamics ; RNA, Viral ; blood ; Viral Load ; Virus Replication

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 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

OBJECTIVETo determine the genetic relationships between different Vibrio cholerae isolates in Shenzhen from 1993 to 2002.

METHODSChromosomal DNA from 60 isolates was digested in seakem gold agrose with restriction enzyme Not I and plugs were then analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns of V. cholerae isolates were clustered using BioNumerics software.

RESULTS39 distinctive PFGE patterns were identified with each pattern having 20 to 30 bands. Most PFGE patterns were divided into cluster A or cluster B.

CONCLUSIONThe closely related pandemic clone clusters of V. cholerae strains did exist in Shenzhen. PFGE of V. cholerae could be used for active surveillance and tracking for cholerae.

China ; epidemiology ; Cholera ; epidemiology ; microbiology ; Electrophoresis, Gel, Pulsed-Field ; methods ; Humans ; Phylogeny ; Vibrio cholerae ; classification ; genetics