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PubMed2003

[Dissolved aluminum and organic carbon in soil solution under six tree stands in Lushan forest ecosystems].

Different depths of soils under 6 tree stands in Lushan Botany Garden were sampled and water-digested at room temperature. The dissolved aluminum and organic carbon were then determined by colorimetry, using 8-hydroxylquilin and TOC Analyzer, respectively. The results indicated that even derived from a naturally identical soil type, the test soils exhibited a diverse solution chemistry, regarding with the Al speciation. The soil solutions under Japanese cedar, giant arborvitae and tea had lower pH values and higher contents of soluble aluminum than those under Giant dogwood, azalea and bamboo. Under giant arborvitae, the lowest pH and the highest content of total soluble aluminum and monomeric aluminum were found in soil solution. There was a significant correlation between soluble aluminum and DOC, which tended to depress the accumulation of toxic monomeric aluminum. The 6 tree stands could be grouped into 2 categories of solution chemistry, according to aluminum mobilization.

Aluminum↗

PubMed2003

[Protective effect of 3,4-oxo-isopropylidene-shikimic acid on vascular endothelial cell injured by hydrogen peroxide].

AIM: To study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on H2O2 (200 mol.L-1, 4 h) injured human umbilical vein endothelial cells (HUVEC). METHODS: Morphological change was observed under microscop. Cell viability was assessed by MTT assay. The release of intracellular lactate dehydrogenase (LDH) and NO was assessed by colorimetry. Radioimmunoassay was used to assess 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). RESULTS: Pretreatment with ISA for 6 h alleviated the morphological damage of H2O2 induced HUVECs. At the concentration of 1-100 mumol.L-1, ISA prevented the inhibitory effect on cell viability induced by H2O2 in dose-dependent manner, but increased the ratio of cell viability from 60.4% to 84.3%. ISA reduced LDH release and increased the level of NO and 6-keto-PGF1 alpha in H2O2 induced HUVECs. CONCLUSION: ISA exerted protective effect on H2O2 injured HUVEC.

6-Ketoprostaglandin F1 alpha↗

PubMed2004

[Prediction of genetic risk for hypertension].

BACKGROUND: Although genetic epidemiological studies have suggested that several genetic variants increase the risk for hypertension, the genes that underlie genetic susceptibility to this condition remain to be identified definitively. Large-scale association studies that examine many gene polymorphisms simultaneously are required to predict genetic risk for hypertension. METHODS AND RESULTS: The population of the present study comprised 1,940 unrelated Japanese individuals, including 1,067 subjects with hypertension (574 men, 493 women) and 873 controls (533 men, 340 women). The genotypes for 33 single nucleotide polymorphisms of 27 candidate genes were determined with a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. Multivariate logistic regression analysis with adjustment for age, body mass index, and the prevalence of smoking, diabetes mellitus, hypercholesterolemia, and hyperuricemia revealed that two polymorphisms (825C -> T in the G protein beta3 subunit gene and 190G -> A in the CC chemokine receptor 2 gene) were significantly associated with hypertension in men and that one polymorphism (-238G -> A in the tumor necrosis factor- alpha gene) was significantly associated with hypertension in women. CONCLUSION: These results suggest that two and one genes may be susceptibility loci for hypertension in Japanese men and women, respectively, and that genotyping of these polymorphisms may prove informative for prediction of the genetic risk for hypertension.

Female↗

PubMed2004

[Effect of melatonin on the expression of nuclear factor-kappa B and airway inflammation in asthmatic rats].

OBJECTIVE: Nuclear factor-kappa B (NF-kappaB) is a critical transcription factor governing the expression of many cytokines that are involved in the pathogenesis of inflammatory diseases, such as asthma and rheumatoid arthritis. Melatonin (MT), a relatively safe and potent antioxidant which has shown efficacy in several chronic inflammatory models, may inhibit the expression of NF-kappaB and therefore might have a therapeutic use in asthma. This study aimed at observing the effect of MT on the expression of NF-kappaB and airway inflammation in a rat model of bronchial asthma. METHODS: Twenty-four male Sprague-Dawley (SD) rats weighing 120 g to 170 g were randomly divided into three experimental groups (8 in each): (1) Asthmatic group: Rats were immunized on day 1 by intraperitoneal injection of 100 mg ovalbumin (OVA) in 1 ml of saline with 100 mg of alu minum hydroxide. From day 15 the animals were challenged with aerosolized OVA (1% in saline) for 20 minutes per day for 7 consecutive days. (2) MT group: OVA-sensitized rats were injected intraperitoneally with 10 mg/kg MT 30 minutes before each OVA challenge. (3) CONTROL GROUP: OVA for inhalation and MT for intraperitoneal injection was replaced with normal saline (NS). Airway responsiveness to aerosolized acetylcholine of 24 rats was detected six hours after the last challenge. Then the rats were lavaged and total and differentiated leukocytes counts in bronchoalveolar lavage fluid (BALF) were performed after staining with Wright-Giemsa staining. At the same time, levels of nitric oxide (NO) in BALF, inducible nitric oxide synthesis (iNOS) and constitute nitric oxide synthesis (cNOS) in the lung tissues were assessed with the use of nitrate reductase and chemical colorimetry, respectively. The expression of NF-kappaB in the lung tissues was observed by means of immunohistochemical staining. RESULTS: (1) After OVA challenge, there was a significant decrease in airway responsiveness, lymphocytes and eosinophils in BALF in MT group compared with asthmatic group (P < 0.01 respectively); (2) There was a significant decrease in amounts of NO(2)(-)/NO(3)(-) in the BALF and levels of iNOS in the lung tissues in MT group comparing with asthmatic group (P < 0.01 respectively); and the levels of iNOS in the lung tissues was correlated positively with NO(2)(-)/NO(3)(-) in the BALF (P < 0.01), but there were no significant differences in activity of cNOS in any of the groups analyzed. (3) There was a significant increase in expression of NF-kappaB in lung tissues in asthmatic group compared with the other groups (P < 0.01), and so was in MT group compared with control group (P < 0.05). CONCLUSIONS: MT could partially inhibit the expression of NF-kappaB and down-regulate the activity of iNOS in lung tissue, decrease the production of NO in BALF. These data suggest that the inhibitory effect of MT probably play a role in decreasing airway hyperresponsiveness and airway inflammation of asthmatic rats model.

Animals↗

PubMed2003

Tracking the elusive antiestrogenic effect of melatonin: a new methodological approach.

OBJECTIVES: Detection of the antiestrogenic effect of melatonin on various breast cancer cell lines and its dependence of the differential expression of estrogen receptors (ERalpha and ERbeta) and melatonin receptors (mt1 and RZRalpha). SETTING AND DESIGN: Dose-response curves of estradiol were determined in 6 different breast cancer cell lines using a colorimetric proliferation assay in the absence or presence of various melatonin concentrations. METHODS: In order to detect the minor growth inhibitory effect of melatonin, a simple yet novel approach was employed: instead of incubating cells at single estradiol-concentrations at increasing melatonin levels, breast cancer cells were grown in microwell-plates for 4 days at increasing concentrations of estradiol (10(-12)M - 10(-10)M) in the absence or presence of melatonin (10(-9)M - 10(-8)M). Cell number was determined using Alamar blue and colorimetry. RT-PCR was performed for the expression of ERalpha, ERbeta, RZRalpha and mt1. RESULTS: Melatonin at concentrations of 10(-9)M and 5 x 10(-9)M shifted the dose-response curves of estradiol to higher concentrations. Responsiveness to melatonin depended on expression of ERalpha but not on ERbeta. mRNA of ERbeta was not detectable in the breast cancer cell lines used. Only small amounts of mt1 transcripts were detectable in MCF-7 cells of one source. In MCF-7 cells transfected with the mt1 gene and in an ovarian cancer cell line mt1 was expressed at significant levels. RZRalpha was expressed in all tested cell lines at different amounts. CONCLUSION: The growth of all ERalpha-positive breast cancer cell lines can be inhibited by melatonin. The effect in most cell lines is weak yet clearly reproducible. RZRalpha clearly contributes to the growth inhibitory effect of melatonin.

Adjuvants, Immunologic↗

PubMed2004

[Effects of Smac gene overexpression on chemotherapeutic sensitivity of gastric cancer cell line MKN-45].

BACKGROUND & OBJECTIVE: Abnormal apoptosis is one of the key factors for drug resistance of neoplasms. Smac, a novel gene involved in regulation of apoptosis,plays an important role in tumor cell apoptosis induced by chemotherapeutic drugs. This study was designed to explore the effects of overexpressed Smac gene on chemotherapeutic sensitivity of gastric cancer cell line. METHODS: By liposome GeneSHUTTLE-40, Smac gene was transfected into gastric cancer cell line MKN-45. Cellular Smac gene expression were determined by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot analysis. Cisplatin (1, 5, 10 microg/ml), mitomycin C (0.1,1,10 microg/ml), and curcumin (10, 20, 40 micromol/L) were selected to treat untransfected and transfected MKN-45 cells. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry. The morphological changes of cancer cells were observed under inverted microscope. Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry. RESULTS: Compared with untransfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly increased (P< 0.01). The growth inhibition rates of MKN-45 cells treated with various concentration of cisplatin, mitomycin C, and curcumin on MKN-45 cells were increased by 10.10%-23.80% (P< 0.01), 10.01%-15.86% (P< 0.01), 11.28%-22.12% (P< 0.01), respectively, with the cells becoming round, obviously refracted, and fragmented. The cellular apoptosis rates were increased by 6.7%-20.2% (P< 0.01), 5.4%-13.2% (P< 0.01), and 10.6%-20.1% (P< 0.01), respectively. CONCLUSION: Transfection of extrinsic Smac gene results in its over-expression in MKN-45 cells,which could increase the chemotherapeutic sensitivity of MKN-45 cells.

Apoptosis↗

PubMed2004

[Correlation between serum p53 antibody and chemosensitivity in patient with esophageal cancer].

BACKGROUND & OBJECTIVE: Studies have revealed that overexpression of p53 protein has close relation to induction of drug resistance in cancer patients and it can be used as a predictor for chemosensitivity of tumor. Recently, it has been found that specific p53 antibody (p53-Ab) presented in the serum of cancer patients with p53 protein overexpression. Furthermore, the presence of serum p53-Ab is closely correlated with p53 protein overexpression. Thus, serum p53-Ab could theoretically be useful in predicting chemosensitivity in cancer patients. In the present study, we investigated whether preoperative serum p53-Ab is correlated with the postoperative chemosensitivity of esophageal cancer, which was analyzed using in vitro drug response assay. METHOD: Serum analysis of p53 antibodies was performed by enzyme-linked immunosorbent assay (ELISA) in 38 patients with esophageal cancer preoperatively, then surgically resected specimens were analyzed for their chemosensitivity to cisdichlorodiammineplatinum (DDP),5-fluorouracil (5-FU), and Adriamycin (ADM) by in vitro drug response assay MTT colorimetry. RESULTS: Serum p53-Ab was present in 47.3% of the patients with esophageal cancer. The positive rate of serum p53-Abs in patients was significantly different from that in healthy blood donors (P< 0.01). The positive rate of serum p53-Ab was 25%(5/20) for the patients at stage I and II, and 72.2%(13/18) for the patients at stage III and IV.A significant correlation between serum p53-Ab positive rate and TNM stage was observed (P< 0.05). The positive rate of serum p53-Ab were 21.5%(3/14), 50%(6/12), 75% (9/12) for the patients with well, moderately,and poorly differentiated tumors, respectively. There was significant correlation between serum p53-Ab positive rate and tumor differentiation (P< 0.05). The chemosensitivity to DDP, 5-FU, and ADM (11.1%,16.1%,and 16.1%, respectively) of the patients with positive serum p53-Ab was significantly lower than that of the patients with negative p53-Ab (60%, 45%, and 35%; P< 0.01,P< 0.05, and P< 0.05, respectively). CONCLUSION: p53-Ab is not only a prognostic marker for patients with esophageal cancer, but also a predictor of their response to chemotherapy.

Adult↗

PubMed2003

[Anergy, apoptosis and decreased MHC class I molecule expression of C57BL/6J mouse CD4+ T cells induced by SEB].

AIM: To study the mechanism of peripheral T cell tolerance induced by superantigen Staphylococus aureus enterotoxin B (SAgSEB). METHODS: Splenocytes from C57BL/6J (B6) mice were stimulated with SEB in-vitro. The proliferation of lymphocytes was determined by MTT colorimetry. The apoptosis of anergic T cells, the cells being in the S and G(0)-G(1) phase and MHC-I (H-2K(b)) expression of T cells were analyzed by flow cytometry (FCM). Apoptosis of anergic T cells was also demonstrated by DNA electrophoresis ladder. RESULTS: SEB could induce strong proliferation of CD4+ T cells and the maximal percentage of CD4+ T cells in S phase appeared on 3rd day after SEB stimulation and then decreased, but the changes of percentage of CD4+ T cells in G(0)-G(1) phase was opposite to that in S phase. CD4+ T cells became anergy on 3rd day and apoptosis of anergic CD4+ T cells appeared on 5th day. Percentage of apoptosis cells increased gradually and could not be reversed by addition of anti-CD3 Ab and ConA. Expression of MHC-I (H-2K(b)) was marginally declined accompanied by CD4+ T cell anergy after SEB exposure. CONCLUSION: SEB-induced tolerance may have relation to anergy and apoptosis of CD4+ T cells, and down-regulation of MHC-I molecules.

Animals↗

PubMed2003

[The effect of calcium ionophore on dendritic cells derived from peripheral blood mononuclear cells].

AIM: To explore the effect of calcium ionophore (CI) on dendritic cells (DC) derived from peripheral blood monocytes. METHODS: Peripheral blood monocytes from healthy donors were treated with 100 microg/L rhGM-CSF, 100 microg/L rhGM-CSF plus (10 microg/L) CI, and 100 microg/L rhGM-CSF plus (100 microg/L) respectively. After cultivation of 40 hours, cellular morphology was observed under light microscope and electron microscope. Surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was used to detect proliferation of autologous T cells. RESULTS: Peripheral blood monocytes treated with 100 microg/L rhGM-CSF plus 100 microg/L CI for 40 hours showed typical morphology of DCs. Simultaneously there was a decrease in CD14 expression and increase in HLA-DR, CD40, CD83 and CD86 expressions on these cells. In addition, PBMCs treated with 100 microg/L rhGM-CSF pass 100 microg/L CI for 40 hrs. Could evidently stimulate proliferation of autologous T cells. CONCLUSION: CI can markedly enhance transformation of peripheral blood monocytes induced by GM-CSF into DCs.

Antigens, CD↗

PubMed2003

[Suspension culture of protocorm-like bodies from the endangered medicinal plant Dendrobium huoshanenese].

OBJECTIVE: To investigate the characteristics of growth, and water-soluble polysaccharide and total alkaloid accumulation in protocom-like bodies (PLBs) of Dendrobium huoshanenese in liquid culture system. METHOD: PLBs were suspended in liquid medium and growth kinetics was analyzed. Water-soluble polysaccharide and total alkaloid content in PLBs were determined by colorimetry. RESULT: PLBs were induced from stem explants of D. huoshanenese regenerants cultured on MS medium supplemented with NAA or NAA and KT at different concentrations. Basal MS medium was suitable for propagation of PLBs. When PLBs were suspended in liquid medium, mumax was 0.044.d-1, t(d) was 15.8 d and the optimum growth time was 4 weeks. Water-soluble polysaccharide and total alkaloid contents in PLBs were 3.75% and 0.0261%, respectively. CONCLUSION: PLBs in liquid culture system show a potential for rapid growth and high metabolite synthesis, which provides possibility for exploiting resources of D. huoshanenese by large scale culture of PLBs.

Alkaloids↗

PubMed2004

[The protective effect of interleukin-1 receptor antagonist on postischemic reperfused myocardium and its possible mechanism].

OBJECTIVE: To observe the dynamic changes of plasma inflammatory cytokine interleukin-1 beta (IL-1 beta) in patients with acute myocardiac infarction (AMI) before and after recanalization of the infarct related artery (IRA) and to observe the effect of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the postischemic reperfused myocardium in experimental rabbits. METHODS: (1) ELISA was used to measure the plasma IL-1 beta of 22 AMI patients, 20 males and 2 females, aged 64 +/- 12, before emergency percutaneous coronary intervention (PCI), and 12 hours and 24 hours after-intervention, and measure the plasma IL-1 beta of 8 healthy controls, 6 males and 2 females, aged 56 +/- 9. (2) Forty rabbits underwent ligation of the left circumflex branch of coronary artery (LCX) for 50 minutes and reperfusion for 4 hours after the ligatures were untied. The rabbits were randomly divided into 4 groups of 10 rabbits to be injected into the left ventricle immediately before the reperfusion with rhIL-1ra 10 mg/kg (group A), 20 mg/kg (group B), or 40 mg/kg (group C), and normal saline (control group) respectively. After reperfusion of 4 hours, the LCX was re-ligated. Evans blue was injected into the left ventricle. 15% KCl was injected intravenously to kill the rabbits. Their hearts were taken out to weigh the non-ischemic, ischemic, and necrotic cardiac muscles so as to calculate the infarct size. The myoperoxidase (MPO) activity was measured by colorimetry. Sections of myocardium were made. The number of apoptotic cardiomyocytes was evaluated by TUNEL method. The apoptotic rate of cardiomyocyte was measured by annexin V method. The DNA expression of myocardium was detected by DNA laddering method. The expressions of Bcl-2 and Bax apoptosis genes were assessed. RESULTS: (1) The average plasma IL-1 beta level of the 22 AMI patients before emergency PCI was significantly higher than that of the controls (28 pg/ml +/- 9 pg/ml vs. 20 pg/ml +/- 11 pg/ml, P < 0.05), and became the highest 12 hours after the intervention (86 pg/ml +/- 14 pg/ml), and the high level lasted to 24 hours after emergency PCI. (2) In the ischemia-reperfusion rabbit model, the infarct size was 47% +/- 7% in the group A, 34% +/- 8% in the group B, 31% +/- 6% in the group C, and 61% +/- 11% in the control group (P < 0.05, 0.01, and 0.01 respectively). The activity of myocardial MPO was 16.6 +/- 3.6 min(-1).g.w.w(-1) in the group A, 10.9 min(-1).g.w.w(-1) +/- 1.9 min(-1).g.w.w(-1) in the group B, 7.8 min(-1).g.w.w(-1) +/- 2.2 min(-1).g.w.w(-1) in the group C, and 20.5 min(-1).g.w.w(-1) +/- 4.5 min(-1).g.w.w(-1) in the control group (P < 0.05, 0.01, and 0.01 respectively). The cardiomyocyte apoptosis evaluated by TUNEL was 38.3 n/HP +/- 7.4 n/HP in the group A, 25.6 n/HP +/- 6.8 n/HP in the group B, 12.2 n/HP +/- 3.3 n/HP in the group C, and 44.4 n/HP +/- 9.5 n/HP in the control group (P < 0.05, and P < 0.01 respectively in comparison between the group B and the control group and between the group C and the control group). The apoptotic rate by annexin V method was 11.6% +/- 2.7% in the group A; 7.7% +/- 2.4% in the group B, 4.7% +/- 1.4% in the group C, and 15.6% +/- 3.5% in the control group (P < 0.05, 0.01, and 0.01 respectively). DNA electrophoresis showed scaling ladder pattern only in the control group. The fluorescent density of the apoptosis gene Bax in myocardium was 24.9 +/- 8.2 in the group A; 15.5 +/- 3.4 in the group B, 10.6 +/- 2.5 in the group C, and 33.3 +/- 9.4 in the control group (P = 0.0298, 0091, and 0052 respectively) and no significant difference in the expression of Bcl-2 was shown among the 4 groups. Myocardial MPO was correlated with cardiomyocyte apoptosis (r = 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05). CONCLUSION: Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-als; 0.86 by TUNEL, P < 0.01; r = 0.75 by Annexin V method, P < 0.05). CONCLUSION: Inflammatory cytokine IL-1 beta is involved in myocardial ischemia-reperfusion injury. With potential therapeutic value in prevention and treatment of ischemia-reperfusion injury to myocardium, rhIL-1ra may reduce myocardial ischemia-reperfusion injury by suppression of cardiomyocytes apoptosis mediated by IL-1.

Acute Disease↗

PubMed2003

[The study of primary culture methods for hippocampal neurons of newborn rats].

AIM: To compare three different methods for neuron culture, so as to provide a culture technique with higher neuron purity and survival rate. METHODS: Neurons in hippocampal region of newborn SD rats were cultured by common culture method, cytosine arabinoside (Ara-c)-supplementing method, Ara-c and nerve growth factor (NGF)-supplementing method. Then morphology of neurons was observed under microscope. The survival rates of the neurons at different culture times were compared by inverted microscope observation and MTT colorimetry. The purity of neurons cultured by 3 methods was detected by immunocytochemical staining. RESULTS: Neurons cultured by Ara-c and NGF-supplementing method grew well. The purity and survival rate of neurons cultured by Ara-c and NGF-supplementing method was highest. CONCLUSION: Ara-c and NGF-supplementing method is a good method for neuron culture.

Animals↗

PubMed2003

[Study of the inhibiting effect of anti-NKG2D polyclonal antibody on cytotoxicities of NK and LAK cells].

AIM: To investigate the effect of anti NKG2D polyclonal antibody(pAb) on cytotoxicities of NK and LAK cells. METHODS: Peripheral blood mononuclear cells(PBMCs) were separated by centrifugation on Ficoll-Hypaque gradients. LAK cells were induced from PBMCs by PHA (10 mg/L) and rhIL-2 (1x10(6)U/L). Then NK cells were sorted by flow cytometry(FCM). The cytotoxicities of NK and LAK cells were analyzed by MTT colorimetry after NKG2D molecule on NK and LAK cells were blocked with anti-NKG2D pAb. RESULTS: FCM analysis proved that both purity and activity of obtained NK cells were high.The anti-NKG2D pAb could inhibit significantly cytotoxicities of NK and LAK cells to K562 and HepG2 cells, for NK cells,having decreased 82.9% and 75.6%, for LAK cells,having decreased 52.8% and 50.2%, respectively.The anti-NKG2D pAb had no effect on cytotoxicities of NK and LAK cells to CNE cells. CONCLUSION: The anti-NKG2D pAb can inhibit cytotoxocities on tumor cells by NK and LAK cells through NKG2D molecule on two effector cells.

Cytotoxicity, Immunologic↗

PubMed2003

[Expression of a novel human interleukin-13 in E.coli and its biological activity assay].

AIM: To express a novel Gln98-deleted human interleukin-13 in E.coli. METHODS: Total RNA was isolated from Jurkat cells costimulated with PHA and ConA. A 358 bp-specific DNA fragment encoding hIL-13 was amplified by semi-nested RT-PCR. DNA sequencing showed that the target DNA was a Gln98-deleted novel splicing of hIL-13. This hIL-13 cDNA and plasmid pBV220 were ligated at BamH I and EcoR I sites to construct the expression vector. After transforming E.coli strain DH5alpha, the expression of the novel splicing hIL-13 gene was induced by shifting culture temperature from 30 degrees Celsius to 42 degrees Celsius. The expression product was then purified by chromatography on Sepharcryl-200 gel column, and the bioactivity was detected by MTT colorimetry on the growth of TF-1 cell line. RESULTS: The novel rhIL-13 was expressed in the form of inclusion bodies. After purification and renaturation, the specific activity of the novel rhIL-13 was 1.6x10(6) IU/mg. CONCLUSION: The novel rhIL-13 with biological activity has been obtained, which lays the foundation for treating cancer and septicemia by the cytokine in future.

DNA, Complementary↗

PubMed2003

[Enhancement effect of IL-2 on the proliferation of cultured mammary carcinoma cell line Bcap-37].

AIM: To investigate the effects of IL-2 on the proliferation of human mammary carcinoma cell line Bcap-37 and explore its possible mechanism. METHODS: Enhancement effect of IL-2 on proliferation of cultured Bcap-37 cells, the IL-2 expression on the cells, the expressions of IL-2Ralpha, beta, gamma mRNAs in the cells, the effect of IL-2 on DNA content at various periods of cell cycle and on Ca(2+) concentration in the cells were detected or observed by MTT colorimetry, immunohistochemical staining, RT-PCR, flow cytometry and laser scanning confocal microscope, respectively. RESULTS: IL-2 of 1x10(5) to 1x10(6) U/L significantly stimulated the proliferation of cultured Bcap-37 cells. IL-2 was secreted and IL-2Ralpha, beta and gamma were expressed by cultured Bcap-37 cells. IL-2(1x10(5) U/L) increased ratio of Bcap-37 cells in G(2) and S phases and decreased the Ca(2+) release from Bcap-37 cells. CONCLUSION: IL-2 significantly enhance the proliferation of mammary carcinoma cell line Bcap-37. The enhancement effect of IL-2 on Bcap-37 cell proliferation is possibly related to the expression of IL-2R and decreased Ca(2+) concentration in the cells.

Cell Cycle↗

PubMed2003

[Purification and characterization of the fusion protein TGF-betaR II/Fc].

AIM: To express the fusion protein TGF-betaR II/Fc in large amounts by using recombinant Bac-TR II baculovirus expression system constructed by our laboratory and to purify and characterize it. Then, to verify whether the fusion protein TGF-betaR II/Fc can be able to block the biological activity of cytokine TGF-beta1. METHODS: The viral titer was determined by plaque forming test. The recombinant baculovirus was amplified by infecting sf9 cells. The fusion protein was purified by FPLC using protein G column. The purified product was analyzed by SDS-PAGE and the amount of target protein calculated by gray scanning. Western blot and sandwich ELISA were used to affirm the expression of the fusion protein. MTT colorimetry was used to test whether the fusion protein can block the inhibition effect of cytokine TGF-beta1 on the growth of L929 cells. It was to verify whether the fusion protein can reduce the fibronectin production in L929 cells accelerated by TGF-beta1 by western blot. RESULTS: The titer of recombinant Bac-TR II baculavirus in the primary culture fluid was 2x10(12) pfu/L. After electrophoresis, gray scanning analysis showed that the target protein accounted for 10 percent of the total protein. Western blot analysis and sandwich ELISA detection proved that the target protein has been expressed. The fusion protein could block the inhibitive effect of cytokine TGF-beta1 on the growth of L929 cells and fibronectin production in L929 cells. CONCLUSION: The fusion protein TGF-betaR II/Fc can inhibit the biological activity of TGF-beta1 in-vitro. This study will be helpful to the mass production of the fusion protein, and will facilitate its further use in the therapy of pulmonary fibrosis.

Blotting, Western↗

PubMed2003

[Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471].

AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry. RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha. CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.

Animals↗

PubMed2003

[The relationship between abnormal apoptosis and Fas expression of thymocytes from patients with myasthenia gravis].

AIM: To explore the roles of abnormalities of thymocytic abnormal apoptosis and its Fas expression in patients with myasthenia gravis (MG) in generation of autoimmune response. METHODS: The thymocytes were isolated and thymus extractive was prepared from fresh thymic tissue excised from MG patients. The proliferation of thymocyes was evaluated by MTT colorimetry. The apoptosis of thymocytes were examined by DNA electrophoresis. Expression of Fas was analyzed by a FACScan flow cytometry. Transcription and mutation of Fas gene were detected by RT-PCR and single-stranded conformation polymorphism ( SSCP) analysis. RESULTS: Thymus extractive could inhibit proliferation of normal thymocytes, while had no influence on the thymocytic proliferation of MG patients. The proliferation of thymocytes of both normal persons and MG patients could be inhibited by thymus extractive in the presence of dexamethasone. The inhibition rates were 58. 33% and 54. 26% respectively. The cocultured thymocytes with dexamethasone showed characteristic apoptosis pattern and the DNA electrophoresis exhibited 'ladder' appearance. The abnormal bands in Fas mRNA of partial MG patients' thymocytes were found by RT-PCR-SSCP analysis. CONCLUSION: Abnormalities of apoptosis and Fas gene expression of MG patients' thymocytes, and Fas gene mutation may be related to the pathogenesis and progression of MG.

Adolescent↗