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At least 937 records · Page 52Linked to original sources

PubMed2004

[Construction, expression and cell growth inhibition of translocating peptide/granzyme B fusion protein gene].

AIM: To investigate the inhibitory effect of translocating peptide/granzyme B fusion protein on cell growth. METHODS: The translocating peptide gene of Pseudomonas exotoxin A (PE) was fused with active granzyme B gene by recombinant PCR to construct PE II-GrBa fusion protein gene. PE II-mGrBa with a mutation of serine to cystein at active center of GrB was used as negative control. The resulting PE II-GrBa and PE II-mGrBa genes were transiently transfected into mammalian cells via lipofectamine mediation. The effects of expression of PE II-GrBa gene on morphology and growth of transfected cells were detected by MTT colorimetry, TUNEL assay and indirect immunofluorescence staining. RESULTS: Transient expression of PE II-GrBa resulted in cytoskeleton abnormality, cell growth inhibition, and apoptosis in some cells. CONCLUSION: Expression of PE II-GrBa fusion protein can inhibit cell growth.

Apoptosis↗

PubMed2004

[Screening of antagonist peptide of BLyS from C7C phage display peptide library].

AIM: To screen an antagonist peptide of BLyS from C7C phage display peptide library. METHODS: C7C phage display peptide library was screened with BLyS. Indirect ELISA, competitive ELISA and MTT colorimetry were used to identify positive phage clones. RESULTS: After 3 rounds of screening, the gradual increase of the ratio of output to input and specific enrichment had been achieved. Two phage clones that could inhibit the BLyS activity were identified. CONCLUSION: Two phage display antagonist peptides of BLyS were obtained.

B-Cell Activating Factor↗

PubMed2004

[Cloning, expression, renaturation and purification of soluble hSCF].

AIM: To construct an expression vector for soluble recombinant human stem cell factor (rhSCF) gene, and optimize culture conditions for high-level expression of rhSCF in E.coli. METHODS: hSCF cDNA was amplified by PCR with total cDNA from human lymph node as a template, followed by cloning into pMD18-T vector. The 5' terminal of the hSCF cDNA was modified by degenerative PCR to obtain high-level expression in E.coli. The biological activity of the refolded rhSCF, purified with high performance hydrophobic interaction chromatography, was examined by MTT colorimetry. RESULTS: hSCF cDNA was amplified and cloned, and was inserted into an E.coli expression vector pBV220. The expressed rhSCF accounted for about 20% of total bacterial proteins and reached 40% of total bacterial proteins under optimal culture conditions. The expressed rhSCF appeared in bacterial lysates in the form inclusion body. The rhSCF with biological activity was obtained after solubilization of the inclusion body with 8 mol/L urea or 7 mol/L guanidine chloride, followed by preliminary refolding and purification. CONCLUSION: hSCF was cloned and expressed in E.coli successfully. The E.coli strain expressing rhSCF can be used to produce rhSCF with biological activity on large-scale production.

Cell Line, Tumor↗

PubMed2004

[The induction of differentiation into dendritic cells from HL-60 cells by calcium ionophore].

AIM: To explore whether the promyelocytic leukemia cell line HL-60 may differentiate into activated dendritic cells (DCs) by A23187, a calcium ionophore. METHODS: The HL-60 cells were cultured in common medium alone or with various concentrations of A23187 (25-1 600 microg/L) and rhGM-CSF (100 microg/L). After culture for 24-96 hours, the cellular morphological change was observed under light microscope and electron microscope. Surface makers on treated HL-60 cells were analyzed by flow cytometry. The proliferation of allogeneic human T cells was detected by MTT colorimetry. RESULTS: Under the condition of a suitable dose (200 microg/L) of A23187 treatment of HL-60 cells for 24 hours, the expression of CD83 molecule, a characteristic marker on DCs, was highest. The typical dendritic outgrowth appeared at a time when HL-60 cells were treated with A23187 for 72 hours. However, when HL-60 cells were treated with A23187 for 96 hours, the expressions of CD80 (B7.1), CD86 (B7.2), MHC-class II molecule and CD54 on HL-60 cells reached peak, and marked activation of allogeneic T cells occurred. CONCLUSION: Calcium ionophore A23187 can induce the HL-60 cells to differentiate into activated DCs-like cells.

Antigens, CD↗

PubMed2004

Impact of gestational diabetes on lipid profiling and indices of oxidative stress in maternal and cord plasma.

OBJECTIVE: To study the effect of gestational diabetes mellitus (GDM) on indices of oxidative stress and lipid profiles in maternal and cord blood samples. METHODS: Blood samples were collected from 40 normal pregnant women and 46 women with GDM during the period 1998 through to 1999 at the Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia. The GDM patients were subdivided into 2 groups: patients receiving insulin treatment (GDM-I, N=19) and patients under control diet (GDM-D, N=27). Plasma a- and y-tocopherols were estimated by high-performance liquid chromatography, whereas malondialdehyde (MDA) was analyzed by fluorometry. Serum lipids (low density lipoprotein, high density lipoprotein, total cholesterol, triglycerides, and total lipids) were determined by enzymatic colorimetry using automated clinical analyzer. RESULTS: The results of lipid profiles in maternal serum showed no significant difference between GDM patients and controls; however, all the lipid constituents except total cholesterol were significantly reduced in the cord blood of GDM patients as compared to control subjects. a-tocopherol levels in the maternal plasma were not significantly different among the 3 groups, whereas, cord plasma a-tocopherol was significantly decreased in both GDM-D and GDM-I. Maternal y-tocopherol was found to be significantly increased in GDM-D and only insignificantly increased in GDM-I, but the cord y-tocopherol showed no appreciable changes. The level of MDA was 3-fold higher in maternal plasma as compared to cord plasma. However, neither the maternal plasma nor cord plasma showed significant differences in MDA levels between GDM patients and normal pregnant women. CONCLUSION: A significant depletion of a-tocopherol in the cord blood of GDM patients is indicative of a possible oxidative stress in their fetuses. Further studies are warranted to examine a wider range of biochemical parameters to evaluate the potential risks of oxidative damage.

Adult↗

PubMed2004

[Immunosuppressive activity of exopolysaccharide from Paecilomyces gunnii].

OBJECTIVE: To investigate bioactivities of Paecilomyces gunnii exopolysaccharide (PGEP). METHODS: The effects of PGEP on mice spleen lymphocytes proliferation, mice peritoneal macrophage (PMphi) phagocytosis and cytotoxin T lymphocytes (CTL) activity were studied by MTT method, neutral red colorimetry, respectively. RESULTS: In vitro, mice spleen lymphocytes proliferation, mice peritoneal macrophage (PMphi) phagocytosis of neutral red and CTL activity were significant suppressed by PGEP, especially in 10 microg/ml. CONCLUSION: PGEP had the immunosuppressive activities.

Animals↗

PubMed2004

[Dynamic changes of enzymes activities and growth factors contents in human colostrum].

OBJECTIVE: To study the dynamic changes of the activities of superoxide dismutase (SOD), amylase (AM) and alkaline phosphatase (ALP) and of the contents of epithelium growth factors (EGF) and insulin growth factors (IGF-1) in human colostrum (lactation 1 - 7 days postpartum). METHODS: A total of 118 samples of human colostrum were taken from 20 healthy women postpartum. The activities of SOD, AM and ALP in human colostrum were determined by methods of pyrogallol autoxidation, colorimetry of iodine-starch and pyrocatechol phosphate respectively. EGF and IGF-1 contents in human colostrum were measured by radioimmunoassay. RESULTS: The SOD activity of human colostrum increased slightly during lactation 1 - 4 days postpartum, from (18.7 +/- 2.2) kU/L to (22.5 +/- 2.9) kU/L, then it tended to decrease, being (11.2 +/- 2.1) kU/L on the 7th day. The SOD activities in lactation 1 - 5 days postpartum were significantly higher than that on day 7 (P < 0.05 or P < 0.01). AM and ALP activities of human colostrum gradually decreased within a week postpartum. AM activity declined from (155 +/- 12) kU/L to (17 +/- 4) kU/L. ALP activity declined from (105 +/- 17) kU/L to (54 +/- 9) kU/L. AM activities in lactation 1 - 5 days postpartum were significantly higher than that on day 7 (P < 0.05 or P < 0.01); and ALP activity on the first lactation day was significantly higher than that on day 7 (P < 0.05). EGF and IGF-1 concentrations in human colostrum quickly declined during lactation 1 - 4 days postpartum. EGF concentration decreased from (105 +/- 11) micro g/L to (40 +/- 5) micro g/L, and IGF-1 concentration decreased from (25.9 +/- 2.7) micro g/L to (5.6 +/- 1.3) micro g/L. Both concentrations altered slightly during lactation 4 - 7 days postpartum. EGF concentrations in lactation 1 - 3 days postpartum were remarkably higher than that on day 7 (P < 0.05 or P < 0.01); IGF-1 concentration on the first lactation day was significantly higher than that on day 7 (P < 0.01). CONCLUSIONS: Human colostrum contains SOD, AM and ALP with high activities, and is also rich in EGF and IGF-1. All of these are indispensable to normal growth and development of infants. Breast-feeding should be emphasized and encouraged for infants.

Adult↗

PubMed2004

[Analysis of the relative etiology of non-obstructive azoospermia].

OBJECTIVE: To investigate the levels and changes of sexual hormone in serum and seminal plasma, and the epidermal growth factor (EGF), alpha-glucosidase, and fructose in the seminal plasma of non-obstructive azoospermic patients, and to assess the significance of the analysis of their chromosomes. METHODS: Thirty-six non-obstructive azoospermic males and 26 normal males were chosen. The results in reproductive endocrine and genetics obtained by radioimmunoassay, colorimetry, and chromosomes G and C banding assay were compared and analyzed to explore into the causes of infertility. RESULTS: The levels of FSH, LH, PRL and E2 in the serum of the non-obstructive azoospermic patients were higher than those of the control group, but the T level had no significant difference between the two groups. There were no obvious differences in the sexual hormones except in FSH of the seminal plasma. There were 2 cases of abnormal sex chromosomes, 2 cases of big Y chromosomes and 7 cases of small Y chromosomes in the non-obstructive azoospermic patients. No correlation was found between EGF and fructose in the seminal plasma. CONCLUSION: The level of sexual hormone in serum, alpha-glucosidase in seminal plasma and abnormal chromosomes might be closely related to non-obstructive azoospermia.

Adult↗

PubMed2004

[Retrovirus-mediated IL-18 gene expression in rat C6 glioma cell line].

AIM: To set up rat C6 glioma cell line C6/IL-18 expressing IL-18 gene and explore the effect of IL-18 on the growth of C6 cells. METHODS: The IL-18 gene was transferred into the C6 cells by a retrovirus vector. After screening with G418, the C6/IL-18 cells were obtained. IL-18 mRNA expression in C6/IL-18 cells was detected with RT-PCR. The expression of IL-18 protein was detected by flow cytometry and immunocytochemical staining. In order to analyze the activity of the expressed IL-18 protein, ELISA was used to detect the ability to secrete IFN-gamma by rat splenocytes induced with the culture supernatant of C6/IL-18 cells. The in-vitro proliferation of C6/IL-18 cells was detected by MTT colorimetry and flow cytometry. The rat model was used to observe the tumorigenic activity of the C6/IL-18 cells. RESULTS: IL-18 mRNA and protein were stably expressed in C6/IL-18 cells. The culture supernatant of C6/IL-18 cells induced secretion of IFN-gamma by rat splenocytes. At the same time, the proliferation rate and in-vivo tumorigenicity of C6/IL-18 cells were markedly reduced as compared with parental C6 cells. CONCLUSION: Exogenous IL-18 gene can inhibit the proliferation and in-vivo tumorigenicity of C6 cells.

Animals↗

PubMed2004

[Signal transduction in the differentiation of human peripheral blood mononucleocytes towards dendritic cells induced by calcium ionophore].

AIM: To explore the intracellular signal transduction pathway in the differentiation of human peripheral blood mononucleocytes (PBMCs) towards dendritic cells (DCs) induced by calcium ionophore (CI). METHODS: PBMCs isolated from a healthy donor were cultured with A23187 plus rhGM-CSF, 100 microg/L each. In some experiments, PBMCs were cultured for 30 minutes with W-7 (10 micromol/L), CsA(0.5 mg/L) or KT5926(1 micromol/L) before addition of rhGM-CSF and A23187. After culture for 40 hours, morphological change of the cells were observed under phase contrast microscope; surface markers on treated PBMCs were analyzed by flow cytometry; the proliferation of allogeneic human T cells stimulated by the treated PBMCs was detected by MTT colorimetry. RESULTS: PBMCs of the healthy donor cocultured with rhGM-CSF plus CI for 40 hours had the typical morphology of DCs, with decreased CD14 expression, and increased CD83, CD80 and CD86 expressions. The proliferation of allogeneic T cells stimulated by PBMCs treated with A23187 plus rhGM-CSF was strengthened. But the morphological changes, surface marker expressions and the ability to enhancing proliferation of allogeneic T cells were inhibited to different degrees by W-7, CsA or KT5926. CONCLUSION: The differentiation of PBMCs towards DCs by CI may be modulated by Ca (2+)/calmodulin and multiple signal transduction pathways downstream.

Antigens, CD↗

PubMed2004

[Eukaryotic expression of anti-CD3 single chain Fv antibody gene and the characterization of its bioactivities].

AIM: To express anti-CD3 scFv in Hela cells and investigate its biological activity. METHODS: DNA fragment encoding anti-CD3 scFv was inserted into eukaryotic expression vector pDisplay. The recombinant expression vector was sequenced and then transfected into Hela cells by electroporation method. The expression of anti-CD3 scFv was identified by in situ hybridization. In-vitro T lymphocyte activation was then detected by (3)H-TdR incoporation method. Anti-CD3 scFv gene-transfected Hela cells were co-cultured with T cells and cytotoxicity was measured by MTT colorimetry. RESULTS: Anti-CD3 scFv gene was correctly inserted into pDisplay and expressed in Hela cells. The secreted anti-CD3 scFv was able to activate T lymphocytes in the presence of anti-CD28 mAb. Cytotoxicity could be observed when anti-CD3 scFv gene-transfected Hela cells were mixed and co-cultured with T lymphocytes. CONCLUSION: Anti-CD3 scFv expressed by Hela cells can activate T lymphocytes.

Antibodies, Monoclonal↗

PubMed2004

[Soluble expression of recombinant immunotoxin against human bladder carcinoma and its anti-tumor activity].

AIM: To construct soluble expression vector of the recombinant immunotoxin against human bladder carcinoma and express and purify the immunotoxin with high biological activity. METHODS: The gene fragment of the immunotoxin containing the genes encoding the humanized single-chain antibody against human bladder carcinoma and the truncated and modified form of Pseudomonas exotoxin(PE) named PE38/KDEL was digested from the plasmid pABDIT and inserted into the expression vector pTMFK containing the FkpA gene. The recombinant plasmid was used to transform the E.coli strain BL21(DE3)-Star and the immunotoxin was co-expressed with the FkpA which served as the folding assistant factor. The immunotoxin was purified through Ni-NTA agarose and anti-PEAb Sepharose 4B columns. The binding activity of the immunotoxin to the antigen on BIU-87 cells was detected by ELISA. The specific cytotoxic effect of the immunotoxin against BIU-87 cells was assessed by MTT colorimetry. RESULTS: The immunotoxin was over-expressed in soluble form in E.coli. The immunotoxin showed good binding activity to the membrane antigen on BIU-87 cells. The result of MTT assay demonstrated that purified recombinant immunotoxin could specifically kill BIU-87 cells. CONCLUSION: The immunotoxin was obtained with specific cytotoxicity to human bladder cancer cells, which lays the foundation for the further application of the immunotoxin in the target therapy of human bladder carcinoma.

ADP Ribose Transferases↗

PubMed2004

[Changes of signal transducer and transcriptional activator 5 (STAT5) in proliferative splenocytes from asthma mice induced by ovalbumin].

AIM: To investigate the changes of signal transducer and transcriptional activator 5 (STAT5) in proliferative splenocyte of asthma mice. METHODS: BALB/c mice were induced to develop asthma by intraperitoneal injection and then nebulized aspiration of OVA. The splenocytes were isolated, and the proliferation of the splenocytes was detected by MTT colorimetry. The phosphorylation of STAT5 and its location were examined by double immunohistochemical staining and the binding of STAT5 with DNA was determined by electrophoretic mobility shift assay (EMSA). RESULTS: OVA could pronouncedly induce the splenocyte proliferation of asthma mice in dose-dependent manner. Stimulation with OVA for 1 and 3 hours could lead to phosphorylation of STAT5 in splenocytes from asthma mice and increase the binding of STAT5 with DNA, suggesting that STAT5 signal pathway plays an important role in splenocyte proliferation of asthma mice induced with OVA.

Animals↗

PubMed2004

[Establishment of Hela cell line inducibly expressing human active granzyme B].

AIM: To construct inducible expression vector for human granzyme B gene and express it in Hela cell line. METHODS: Human active granzyme B gene was obtained by PCR and cloned into the inducible expression vector pIND. The resulting expression vector, together with a helper plasmid pVgRXR, was stably transfected into Hela cells using Lipofectamine 2000. The transfected cells were selected in medium containing G418 and zeocin. The resistant cells were induced with ponasterone A, and the optimal concentration of ponasterone A and time of induction were determined by immunocytochemical staining. Then the effects of the expressed granzyme protein on Hela cells were detected by MTT colorimetry and cytoskeletal staining. RESULTS: The Hela cells that inducibly expressed human active granzyme B were obtained. Induction with 30 micromol/L ponasterone A for 5 days caused the strongest expression of granzyme B. The induced cells appeared as either multinucleate giant cells or pyknotic small cells, and their growth was inhibited. Further analysis demonstrated cytoskeletal abnormality of multinucleate giant cells. CONCLUSION: The establishment of inducible expression system for active granzyme B lays the foundation for further research on biological function of granzyme B.

Cell Proliferation↗

PubMed2004

Characterization of collagen/chitosan films for skin regenerating scaffold.

Various proportions of chitosan/collagen films (70/30% to 95/05%) w/w were prepared and evaluated for its suitability as skin regenerating scaffold. Interactions between chitosan and collagen were studied using Fourier Transform Infrared spectroscopy (FTIR) and Differential Scanning Colorimetry (DSC). Scanning Electron Microscope (SEM) was used to investigate the morphology of the blend. Mechanical properties were evaluated using a Universal Testing Machine (UTM). The chitosan/collagen films were found to swell proportionally with time until it reaches equilibrium. FTIR spectroscopy indicated no chemical interaction between the components of the blends. DSC data indicated only one peak proving that these two materials are compatible at all proportions investigated. SEM micrographs also indicated good homogeneity between these two materials.

Biocompatible Materials↗

PubMed2004

[The effect of HSV-tk/GCV on hepatic specific damage driven by murine ALB promoter/enhancer].

The recombinant vector (pLLTK), containing murine serum albumin (ALB) gene promoter/enhancer directing the herpes simplex virus thymidine kinase (HSV-tk) gene expression was constructed to study its effect on hepatic cell-specific damage. Firstly,in order to compare the hepatic cell-specific transcriptional activity, three vectors were constructed in which green fluorescent protein (GFP) gene was used as a reporter marker. Vector pLE was driven by murine serum ALB gene promoter,whereas pLLE contained not only murine ALB promoter but also an enhancer located at upstream of the promoter was included. In the meanwhile, the vector pLEL had a murine ALB promoter and enhancer placed at the downstream from GFP. After transfected into Hep-G2 (a human hepatic cell line) and HC-11 (a murine breast epithelial cell line), GFP expression was examined by using fluorescence microscope as well as flow cytometer. Secondly, the vector pLLTK was used to observe the killing effect on Hep-G2 cells. The results demonstrated that ALB promoter/enhancer was able to direct hepatic cell-specific GFP expression. Furthermore, HSV-tk expression in Hep-G2 cells was ganciclovir (GCV)sensitive. After seven days of culture with GCV, pLLTK-transfected Hep-G2 cells showed an obvious cell death (53%) when detected by 3-4,5 dimethylthiozol-2-yl-2, 5-diphenyl tetrazolium bromide colorimetry (MTT) assay. Compared to the untreated group, there were no obvious changes in cellular growth inhibition rate in the Hep-G2 cells transfected with a blank control vector pcDNA3. 1 (only 2% of cells appeared cell death). All these results indicated that the above constructs were hepatocyte-specific. It therefore paves a way for further creating a liver-specific damage animal model by transgenic approach using HSV-tk gene expression driven by the ALB promoter/enhancer.

Animals↗

PubMed2004

[Soluble PD-1 can augment anti-tumor immunity induced by HSP70-peptide complex in tumor-bearing mice].

AIM: To explore the effect of soluble PD-1 (sPD-1) on the anti-tumor immunity induced by HSP70-peptide. METHODS: BALB/c mice were immunized with HSP70-peptide, then the cytotoxic activity of splenocytes was detected by MTT colorimetry. Expression of PD-1 and its ligand on splenocytes was determined by semi-quantitative RT-PCR. For some mice, sPD-1 plasmid was injected alone or in combination with HSP70-peptide and then the tumor weight and cytotoxic activity of splenocytes were measured. RESULTS: sPD-1 alone had anti-tumor effect. HSP70-peptide complex not only induced tumor-specific cytotoxic T lymphocytes(CTLs), but also promoted expression of PD-1 and its ligand. The combination of sPD-1 with HSP70-peptide complex induced much more stronger anti-tumor effect as compared with sPD-1 or HSP70-peptide alone. sPD-1 transfection in mice could enhance the cytotoxic activity of splenocytes from immunized mice. CONCLUSION: sPD-1 enhanced the anti-tumor efficacy of HSP70-peptide.

Animals↗

PubMed2004

[The role of Angelica polysaccharides in inducing effector molecule release by peritoneal macrophages].

AIM: To observe the effect of Angelica polysaccharides on effector molecule production by peritoneal macrophages. METHODS: Macrophages were isolated from the peritoneal cavity of BALB/c mice and the primary culture was performed. MTT colorimetry and spectrophotometry were used to examine the effects of Angelica polysaccharides on the releases of effector molecules, such as nitric oxide(NO), tumor necrosis factor-alpha(TNF-alpha), and reactive oxygen species(ROS) as well as inducible nitric oxide synthase (iNOS) and lysozyme(LSZ) activity by peritoneal macrophages. RESULTS: Angelica polysaccharides could promote the releases of NO, TNF-alpha and ROS from macrophages and improved iNOS and LSZ activities in macrophages. However, Angelica polysaccharides had no direct cytotoxicity to tumor cells, but the cultural supernatant of macrophages cocultured with Angelica polysaccharides could kill L929 cells. CONCLUSION: Angelica polysaccharides can promote the releases of NO, TNF-alpha and ROS by macrophages. Angelica polysaccharides may indirectly play the role of anti-tumors through increased TNF-alpha production by macrophages.

Angelica sinensis↗