[Prokaryotic expression and purification of human histone-1 and its activity detection].

AIM: To construct the prokaryotic expression vector of human histone-1, obtain the purified GST-H1 protein, and detect its activity. METHODS: Human histone-1 coding region was amplified from human mammary cDNA library, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-H1 was transformed into E.coli Rossate. The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis. RESULTS: The DNA fragment of about 650 bp was successfully amplified by PCR, cloned into pGEX-KG, and identified by sequencing. The recombinant protein of about M(r); 52 000 was successfully induced, purified and tested well by Kinase assay. CONCLUSION: The recombinant protein of GST-H1 is obtained successfully, which lay the foundation for further research on cell cycle control.

[Inhibition of the expression of FHL2 by RNA interference].

AIM: To construct the eukaryotic expression vector of small interfering RNA (siRNA) targeting human FHL2 and transfect it into human embryo kidney 293T cells to investigate the silencing effect of FHL2 siRNA on the expression of FHL2 gene. METHODS: Two FHL2 siRNAs were designed and inserted into pSliencer 2.1-U6 neo expression vector. Then human embryo kidney 293T cells were cotransfected with the recombinant plasmids and FLAG-tagged FHL2 expression vector. The silencing effect of FHL2 siRNAs on the expression of FHL2 gene was identified by Western blot. RESULTS: The expression vectors of FHL2 siRNAs were constructed and confirmed by DNA sequencing. Western blot showed that FHL2 siRNAs effectively inhibited expression of FHL2. CONCLUSION: The eukaryotic expression vectors of FHL2 siRNAs are constructed successfully. The siRNAs effectively inhibit the expression of FHL2.

[Expression of EYA2 in non-small cell lang cancer].

OBJECTIVE: To identify the expression of Drosophila Eyes Absent Homologue 2 (EYA2) in non-small cell lung cancer (NSCLC) and to investigate its correlation with clinical parameters. METHODS: 59 fresh specimens of lung cancer and paired normal lung tissue were obtained from 59 NSCLC cases treated in the department of thoracic surgery in our hospital from June 2006 to October 2007. Western blotting and immunohistochemistry were used to assay the specimens with goat anti-human EYA2 polyclone antibody. Clinicopathological parameters were collected and the correlation with EYA2 expression was subsequently analyzed. RESULTS: The expression of EYA2 was detected in cytoplasm and nucleus of the cancer cells, but mostly in cytoplasm. Western blotting and immunohistochemistry showed the expression of EYA2 in NSCLC was increased and correlated with pathological type, but not with gender, age, pTNM stage, histological differentiation and lymph node metastasis. EYA2 expression was significantly up-regulated in adenocarcinoma, while not changed in lung squamous cell carcinoma. CONCLUSION: The results of this study suggest that expression of EYA2 in lung adenocarcinoma is augmented. EYA2 is likely participating in the development of lung adenocarcinoma as a transcriptional activator.

[Construction of human Egr-1 promoter and its response to ionizing radiation in tumor cells].

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by ionizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferase reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48 h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation. mediated gene therapy.

High mobility group box-1 protein acts as a coactivator of nuclear factor of activated T cells-2 in promoting interleukin-2 transcription.

High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825-30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein.

[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[XBP-1 interacts with estrogen receptor alpha (ERalpha)].

Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.

Study on inclusion complex of cyclodextrin with methyl xanthine derivatives by fluorimetry.

The inclusion complexes of beta-cyclodextrin (beta-CD) and HP-beta-cyclodextrin (HP-beta-CD) with caffeine, theophylline and theobromine were investigated by fluorimetry. Various factors affecting the formation of inclusion complexes were discussed in detail including forming time, pH effect and temperature. The results indicate that inclusion process was affected seriously by laying time and pH. The forming time of beta-CD inclusion complexes is much longer than that of HP-beta-CD. The optimum pH range is about 7-12 for caffeine, 8-10 for TP, 10.5-12 for TB. The intensities of their fluorescence increase with the decreasing of temperature. Their maximum excitation wavelengths are all in the range of 280-290 nm. The emission wavelength of caffeine and theophylline are both in the range of 340-360 nm, and that of theobromine is about 325 nm. The fluorescence signals are intensified with the increasing concentration of CD. The stoichiometry of the inclusion complexes of CD with these three methyl xanthine derivatives are all 1:1 and the formation constant are all calculated.

Study on the inclusion complexes of cryptotanshinone with beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin.

The inclusion complexes of beta-cyclodextrin (beta-CD) and HP-beta-cyclodextrin (HP-beta-CD) with a kind of tanshinone, cryptotanshinone (CTan) were investigated by using spectrophotometry. Stable inclusion complexes were established in solution and in solid state and were characterized by UV, IR and 1H NMR spectra, respectively. The optimum pH for inclusion is about 7.5. Stoichiometry of the inclusion complex is 1:1. The stabilities of beta-CD and HP-beta-CD to CTan were in the order: beta-CD

[XBP-1 enhances the transcriptional activity of estrogen receptor alpha].

The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.

[Spatial orientation interaction mechanism between chondroitin sulfate and azure A].

The spatial orientation interaction mechanism of chondroitin sulfate (CS) and azure A (AA) was studied by spectrometry. The maximum binding number (N = 151) and the binding equilibrium constant (K = 5. 24 x 10(4)) were obtained. The molecular binding model of the interaction between AA and CS was established. The influence of the molar ratio of AA/CS, ethanol, hydroxypropyl-beta-cyclodextrin, triton X-100 and NaCl on the spectra of AA-CS complex was investigated. We conclude that the color change of complex depends on not only the electrostatic interaction between AA and CS but also the density of AA binding on CS. And the formation of the absorption peak at 550 nm and the color change of complex result mainly from the hydrophobic interaction between AA and AA binding on CS.

[Study of T lymphocyte rDNA transcription activity in chronic benzene poisoning patients].

OBJECTIVE: To observe the changes in peripheral blood T lymphocyte rDNA transcription activity and to study the significance of immune monitoring for patients with chronic benzene poisoning. METHODS: Venous blood samples were withdrawn from 39 patients with chronic benzene poisoning, 20 patients with malignant disease and 22 healthy controls. Cell culture, argyrophil staining method, I-CLQ cell image analysis system were used in this study. rDNA transcription activity which was expressed by the ratio of integrated area (IA) of nucleolus to that of nucleus, and the ratio of integrated optical density (IOD) of argyrophilic nucleolus to that of argyrophilic nucleus. RESULTS: (1) The value of IA and IOD in chronic benzene poisoning patients (7.95% +/- 1.13% and 7.15% +/- 1.15% respectively) were lower than those in controls (9.59% +/- 1.26% and 8.92% +/- 1.18% respectively), P < 0.01. The value of IA and IOD in chronic moderate benzene poisoning patients (6.54% +/- 0.88%) and (5.47% +/- 0.80%) were lower than those to be observed (7.98% +/- 1.06% and 7.13% +/- 0.96% respectively) as well as in mild poisoning patients (8.19% +/- 1.06% and 7.44% +/- 1.06% respectively), P < 0.05. (2) The value of IA and IOD in malignant group (4.10% +/- 1.50%, 3.67% +/- 1.42%) were much more lower than those in controls and in chronic benzene poisoning patients (P < 0.01). CONCLUSION: rDNA transcription activity may be an index to monitor the cellular immune function of chronic benzene poisoning patients.

Short term effect of Salvia miltiorrhiza in treating rat acetic acid chronic gastric ulcer and long term effect in preventing recurrence.

AIM:To study the short-term effect of Danshen (Salvia miltiorrhiza) on acetic acid induced chronic gastric ulcer in rats and its long-term effect in preventing recurrence.METHODS:Rats with acetic acid-induced gastric ulcer were treated with Danshen and cimetidine for 30 days.Traditional gastric mucosal auto-radiography and (3)H-TdR incorporation into gastric mucosa in vitro were employed to study the effects of Danshen in rat acetic acid-induced chronic gastric ulcer, including ulcer index (UI), ulcer inhibitory rate (IR) and label rate (LR).RESULTS:On the day 5, 30 and 126 of ulcer-making, the UI in the Danshen group was obviously lower than that in the cimetidine group and the control group (42.3 ± 3.9, 3.6 ± 1.2, 4.4 ± 2.3; 49.1 ± 3.6, 5.9 ± 1.4, 9.2 ± 1.3; 61.0 ± 3.8, 8.9 ± 2.5, 12.4 ± 2.4, respectively, P < 0.01), the IR (%) in the Danshen group was obviously higher than that in the cimetidine group (31, 59, 64.8; 19, 33, 26, respectively), and the LR in the Danshen group was obviously higher than that in the cimetidine group and the control group (10.0 ± 0.5,16.2 ± 0.8, 15.0 ± 0.6; 9.0 ± 0.5, 13.9 ± 0.6, 10.8 ± 0.7; 6.5 ± 0.7, 10.1 ± 0.5, 8.0 ± 0.7, respectively, P < 0.01). There was no obvious difference in UI in the Danshen group on day 30 as compared with that on day 126.CONCLUSION:Danshen is effective in promoting ulcer healing and preventing recurrence. The mechanism of action is to strengthen the gastric mucosal barrier and to promote the gastric mucosal cell proliferation along the edge of the ulcer.