Objective To explore the effect of transcription factor E2F1 on the apoptosis of small cell cancer line H446. Methods Plamid vector- mediated E2F1 small hairpin RNA (shRNA) was used to silence E2F1 in H446 cell. RT-PCR and western-blot assay were used to detect the expressions of E2F1 and Bcl-2. The apoptosis rate in H446 cell line was detected by flow cytometry assay. Result E2F1 protein was suppressed in shRNA1-modified H446 cell. Sgnificant difference of the apoptosis was shown between E2F1 shRNA1 group and the other two groups. Additionaly, the expression of Bcl-2 protein increased in E2F1 shRNA1-modified cell line. Conclusions E2F1 is highly expressed in H446 small cell lung cancer cell line. E2F1 promotes apoptosis of H446 through upregulating Bcl-2 expression.

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).

The HIV-1 Rev protein facilitates nuclear export of unspliced and singly spliced viral transcripts containing RRE RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA nuclear export can arrest HIV-1 transcriptional process, which clearly, reveals a target for anti-HIV drug development. In this work, a cell-based assay has been established for screening anti-HIV compounds targeting the Rev-mediated RNA nuclear export. This assay utilized a codon-optimized green fluorescent protein (GFP) as reporter gene, which expression is in a Rev-dependent manner. Any compound that inhibits the Rev-mediated RNA nuclear export is identified by reducing emission of GFP. The Z' score of this model is 0.8220. Three thousands compounds were screened and the positive rate was 9.3% with a cutoff at 50% inhibition. IMB7C7, one of the positive compounds, efficiently inhibits viral production from HIV-1 infected cells.

The highly-pathogenic EV71 strain is the primary cause of mortality in hand-foot-and-mouth disease (HFMD) associated with neurological symptoms, for which no clinically effective drugs or vaccines exist. This study aimed to construct infectious cDNA clones of the EV71 highly-pathogenic strain and the cell-culture adapted strain using a reverse genetics approach. The genomic RNAs of EV71 parent strains were used as the templates for RT-PCR amplification, and then the PCR products that overlapped the full-length genome were connected into the pBR322 vector to produce infectious clones of pEV71 (HP) and pEV71 (CCA), respectively. The results showed that the HP strain propagated much more quickly than the CCA strain. The rescued viruses derived from the infectious clones not only maintained their consistency with their parent strains in terms of genomic sequences, but also retained their respective biological phenotypes. This research will contribute to our understanding of EV71 pathogenesis and the development of novel vaccines against HFMD.
Animals ; Cercopithecus aethiops ; DNA, Complementary ; Enterovirus A, Human ; genetics ; growth & development ; isolation & purification ; pathogenicity ; Hand, Foot and Mouth Disease ; virology ; Humans ; Phylogeny ; Vero Cells ; Virus Cultivation

With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.

BACKGROUNDTo construct human papillomavirus type 18 (HPV18 E6E7) adeno-associated virus (AAV) for studying the role of HPV E6E7 in the development of human cancer.

METHODSHPV18 E6E7 genes were inserted into adeno-associated virus expression vector and then infected 293 cell line. The expression of HPV18 E6E7 genes were confirmed by using RT-PCR/Southern blot assay.

RESULTSThere was HPV18 E6E7 genes in the malignantly transformed cell line. The 293TL cells compared with the parent cells transformed cells grew more rapidly, lost their contact inhibition and formed more and large colonies in soft agar.

CONCLUSIONSHPV18 E6E7 AAV was successfully constructed and could induce malignant transformation. HPV18 E6E7 AAV can be use for studying the immortalization and malignant transformation of human normal epithelial cells.

Cell Line ; Cell Transformation, Neoplastic ; Cell Transformation, Viral ; DNA, Viral ; analysis ; DNA-Binding Proteins ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; virology ; Fetus ; Humans ; Kidney ; cytology ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Polymerase Chain Reaction

OBJECTIVETo evaluate the clinical and radiographic results of total lumbar disc replacement with SB Charité III prosthesis.

METHODSFrom Dec 1999 to Dec 2006, total lumbar disc replacement with SB Charité III prosthesis was performed in 65 patients affected with degenerative lumbar disc disorders. Among these patients, 48 (52 prosthesis) were followed up for more than two years (from 2.0 to 7.5 years). There were 22 males and 26 females with an average age of 43 years old (from 36 to 58 years). The diagnosis was lumbar disc herniation with low back pain in 34 patients, discogenic low back pain in 9 patients and failed lumbar disc surgery in 5 patients. All patients underwent standard anterior procedure under general anesthesia. One level replacement was done in 44 patients (L3,4 in 3, L4,5 in 23 and L5-S1 in 18), and two level procedures in 4 patients (L3,4/L4,5 in 1 and L4,5/L5-S1 in 3). Clinical and radiographic results of these patients were evaluated at each follow-up time (1, 3, 6, 12, 24 months after operation and the latest).

RESULTSThe average visual analogue scales score for pain was 9.3 before operation, changed to 4.3 one month after operation, further declined to 2.6 two years after operation and finally to 1.8 at the latest follow-up evaluation. Meanwhile, the average Oswestry Disability Index was 45.8 before operation, 28.6 one month after operation, 12.5 two years after operation and 8.2 at the latest followup evaluation. All operated levels but one maintained mobile and there was no significant loss of range of motion observed. Complications such as implant dislocation or significant subsidence of the prosthesis occurred in none case of this group. All patients but one (98%) were satisfied with the surgery at the latest follow-up evaluation.

CONCLUSIONSTotal lumbar disc replacement is an effective method for the treatment of degenerative disc disorders. Its long-term outcome remains to be verified.

Adult ; Arthroplasty, Replacement ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc ; surgery ; Joint Prosthesis ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

BST-2 plays an important role in host innate immune response via inhibiting the release of HIV-1. HIV-1 accessory protein Vpu can interact with BST-2 through its transmembrane domains, degrade BST-2, and decrease BST-2 that are transported to the cell surface, thus anti-virus function of BST-2 is antagonized. In our study, we constructed plasmid RB connecting Rluc to the N-termimal of BST-2, and plasmid VE connecting EYFP to the C-terminal of Vpu. The two fusion proteins were co-expressed in 293 cells, and the interaction between the two proteins was detected via BRET method. And we further established a stable 293 cell line of dual-expression. By using BRET method, and the interaction between BST-2 and Vpu transmembrane domain as the target, a high-throughput screening assay was created that was expected to seek novel interaction inhibitors.
Antigens, CD ; chemistry ; genetics ; metabolism ; Cell Line ; GPI-Linked Proteins ; chemistry ; genetics ; metabolism ; HIV Infections ; genetics ; metabolism ; virology ; HIV-1 ; genetics ; metabolism ; High-Throughput Screening Assays ; methods ; Human Immunodeficiency Virus Proteins ; genetics ; metabolism ; Humans ; Protein Binding ; Protein Structure, Tertiary ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism

The HIV-1 Rev protein facilitates nuclear export of unspliced and singly spliced viral transcripts containing RRE RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA nuclear export can arrest HIV-1 transcriptional process, which clearly, reveals a target for anti-HIV drug development. In this work, a cell-based assay has been established for screening anti-HIV compounds targeting the Rev-mediated RNA nuclear export. This assay utilized a codon-optimized green fluorescent protein (GFP) as reporter gene, which expression is in a Rev-dependent manner. Any compound that inhibits the Rev-mediated RNA nuclear export is identified by reducing emission of GFP. The Z' score of this model is 0.8220. Three thousands compounds were screened and the positive rate was 9.3% with a cutoff at 50% inhibition. IMB7C7, one of the positive compounds, efficiently inhibits viral production from HIV-1 infected cells.
Active Transport, Cell Nucleus ; drug effects ; Anti-HIV Agents ; pharmacology ; Cell Nucleus ; metabolism ; Codon ; Fatty Acids, Unsaturated ; pharmacology ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; HEK293 Cells ; HIV-1 ; drug effects ; genetics ; High-Throughput Screening Assays ; Humans ; Karyopherins ; genetics ; metabolism ; RNA, Viral ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Transfection ; Virus Replication ; drug effects ; rev Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Bioluminescence Resonance Energy Transfer Techniques ; methods ; Fusion Proteins, gag-pol ; genetics ; metabolism ; HEK293 Cells ; HIV Protease ; metabolism ; physiology ; HIV-1 ; enzymology ; High-Throughput Screening Assays ; methods ; Humans ; Plasmids ; genetics ; Protein Precursors ; metabolism ; physiology ; Pyridazines ; pharmacology ; Transfection ; Virion ; growth & development ; Virus Assembly ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism