Therapeutic Role for TSP-2 Antibody in a Murine Asthma Model.

BACKGROUND: Specific immunotherapy, including agonists for Toll-like receptor 2 (TLR2), have been shown to protect from allergies and to have a high immunomodulatory capacity. METHODS: A new antibody, TSP-2, reactive against an epitope of the extracellular domain of TLR2, was identified. The effect of the antibody on dendritic cells was assessed by immunohistochemistry, Western blot, and flow cytometric analysis. The effect of TSP-2 in a murine asthma model induced with ovalbumin (OVA) was assessed. The model is a form of airway hyperresponsiveness (AHR) and was analyzed by whole-body plethysmography, the measurement of Th1/Th2 cytokines in bronchial alveolar lavage fluid (BALF) and serum by ELISA, and the CCK-8 assay for lymphocyte proliferation. The effect of TSP-2 on the maturation of bone marrow-derived dendritic cells (BMDCs) was assessed by flow cytometric analysis. RESULTS: TSP-2 promoted the maturation of dendritic cells and the proliferation of lymphocyte in vitro and in vivo. The effect of TSP-2 on T helper 1 (Th1)/Th2 cytokine secretion was slightly more powerful than that of Pam3CSK4. TSP-2 antibody reduced AHR and OVA-specific IgE levels in allergic asthma. TSP-2 antibody also reduced lung inflammation and decreased leukocyte numbers in an OVA-sensitized and challenged asthma model. TSP-2 antibody increased OVA-stimulated I-A, CD80, CD86, and MHC-II levels on BMDCs. CONCLUSIONS: This study identifies a novel therapeutic strategy for AHR, which uses antibodies reactive against TLR2. It also provides theoretical evidence for the control of allergic asthma by targeting TLR2.

[Establishment of molecular beacon real-time PCR for detecting p53 gene single nucleotide polymorphism].

OBJECTIVE: To establish a method based on molecular beacon real-time PCR for detecting single nucleotide polymorphisms (SNP) in codon 72 of scar-related p53 gene. METHODS: Two fluorescence-labeled molecular beacon probes were synthesized targeting CCC/CGC SNP of p53 codon 72. The genomic DNA was extracted from the peripheral blood of 28 patients with keloid, and the CCC/CGC SNP of P53 gene codon 72 were assayed with molecular beacon real-time PCR. The results of SNP typing were compared with the results of reverse dot hybridization and confirmed by direct DNA sequencing. RESULTS: The goodness of fit of this method was 100% in comparison with direct DNA sequencing, higher than that of reverse dot hybridization. CONCLUSION: Molecular beacon real-time PCR is suitable for rapid clinical detection of SNPs in p53 gene.

[Effect of metformin on apoptosis of renal cell carcinoma cells in vitro and its mechanisms].

OBJECTIVE: To evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms. METHODS: Fluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9. RESULTS: Metformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin. CONCLUSION: Metformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.

[Ultracytochemical observation of the intracellular localization of H+-adenosine triphosphatase].

OBJECTIVE: To observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles. METHODS: The localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods. RESULTS: H(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats. CONCLUSION: This finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

Metformin induces G1 cell cycle arrest and inhibits cell proliferation in nasopharyngeal carcinoma cells.

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on nuclear factor-kappa B and cytokine expression in the intestine of septic mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the expression of nuclear factor-kappa B (NF-κB) and cytokines in the intestinal tissue of septic mice. METHODS: Male BALB/c mice were randomly divided into 4 groups, namely the sham-operated group, model group, TSP-2 treatment group and rabbit IgG treatment group. Sepsis was induced by cecal ligation and puncture (CLP), and at 6, 12 or 24 h after the operation, the ileal tissues were harvested from the mice for HE staining. NF-κB expression was detected with immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expressions were detected with qRT-PCR and their protein expressions by ELISA. RESULTS: The NF-κB expression in the intestinal tissue significantly increased in the model group as compared with that in the sham- operated group, and decreased after TSP-2 treatment. The model group also showed significantly increased expression levels of TNF-α and IL-6 mRNA and protein in the intestinal tissue (P<0.05), which were lowered by TSP-2 (P<0.05) but not by rabbit IgG treatment (P>0.05). CONCLUSION: The TSP-2 antibody can protect the intestine and delay the development of sepsis by inhibiting NF-κB activation and down-regulating TNF-α and IL-6 expressions in mice.

A primer design strategy for PCR amplification of GC-rich DNA sequences.

OBJECTIVES: To establish a primer design method for amplification of GC-rich DNA sequences. DESIGN AND METHODS: A group of 15 pairs of primers with higher T(m) (>79.7°C) and lower level ΔT(m) (<1°C) were designed to amplify GC-rich sequences (66.0%-84.0%). The statistical analysis of primer parameters and GC content of PCR products was performed and compared with literatures. Other control experiments were conducted using shortened primers for GC-rich PCR amplifications in this study, and the statistical analysis of shortened primer parameters and GC content of PCR products was performed compared with primers not shortened. A group of 26 pairs of primers were designed to test the applicability of this primer designing strategy in amplifications of non-GC-rich sequences (35.2%-53.5%). RESULTS: All the DNA sequences in this study were successfully amplified. Statistical analyses show that the T(m) and ΔT(m) were the main factors influencing amplifications. CONCLUSIONS: This primer designing strategy offered a perfect tool for amplification of GC-rich sequences. It proves that the secondary structures cannot be formed at higher annealing temperature conditions (>65°C), and we can overcome this difficulty easily by designing primers and using higher annealing temperature.

γ-secretase inhibitor-I enhances radiosensitivity of glioblastoma cell lines by depleting CD133+ tumor cells.

BACKGROUND AND AIMS: Glioblastoma is a deadly primary brain tumor with great resistance to radiotherapy. To reverse its radioresistance is important for improving prognosis. Gamma-secretase inhibitors (GSI) have been proven to have anti-tumor effects, yet the knowledge of their influences on glioblastomas is still limited. METHODS: The cytotoxic effects of GSI-I (a tripeptide GSI) on glioblastoma cell lines U87 and U251 were assessed by MTT assay, and the low concentration that did not induce significant cell death was determined. The in vitro radiosensitization effects of this low concentration of GSI-I were evaluated by cell colony forming assays. The CD133+ cell fractions before and after radiation with or without treatment of GSI-I were analyzed by flow cytometry. Then CD133+ and CD133- glioblastoma cells were sorted by magnetic activated cell sorting (MACS), and the radiosensitization effects of GSI-I on the two cell subtypes were investigated separately. Finally, the cytotoxic effects of GSI-I on CD133+and CD133- glioblastoma cells were examined, respectively, and the expression of the Notch pathway components between the two cell subtypes were compared. In addition, the anti-tumor effects of GSI-I were confirmed by in vivo experiments. RESULTS: GSI-I at a low concentration sensitized U87 and U251 cells to radiation by depletion of radioresistant CD133+ cells. CD133+ U87/U251 cells displayed preferential sensitivity to low concentrations of GSI-I, which may be related to the higher expression of the Notch signaling pathway in these cells. CONCLUSIONS: Combining GSI-I with radiotherapy may represent a promising strategy for treating radioresistant gliomas.

[Progesterone promotes the proliferation and migration of cultured breast cancer cells].

OBJECTIVE: To investigate the effects of progesterone on the growth and migration of breast cancer cells. METHODS: MCF-7 and T-47D cells were cultured in DMEM and stimulated with 100 nmol/L progesterone for 48 h, and the cell proliferation was evaluated by MTT assay, cell migration by wound-healing assay and E-catherin expression by Western blotting. RESULTS: Progesterone stimulated the cell proliferation and migration and down-regulated the expression of E-catherin in both MCF-7 and T-47D cells. CONCLUSIONS: Progesterone stimulates the cell proliferation and migration of cultured breast cancer cells, suggesting the clinical significance of anti-progesterone therapy in breast cancer.

[Positional cloning of a novel allele of zebrafish cloche mutant].

OBJECTIVE: To perform the genetic identification of cloche(172) mutant zebrafish. METHODS: The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to treat the AB stain male fish. Large-scale forward genetic screening was carried out to search for lyC-deficient zebrafish mutant by WISH. The morphology changes of the embryos at 3 days postfertilization (3dpf) stage were observed and the cloche(172) gene was identified by mapping and complementation test. RESULTS: We selected 4 lyC-deficient zebrafish by WISH. cloche(172) mutant showed morphological changes similar to cloche mutant in 3dpf stage. One fourth of the embryos showed cloche phenotype as found in complementation test, and the cloche(172) gene was mapped on the telomere of zebrafish 13 chromosome where cloche gene was located. Numerous red blood cells were observed in the cloche(172) mutant, while only a few cells were found in the cloche mutant in the tail region by o-dianisdine staining. CONCLUSION: cloche(172) gene which is responsible for the phenotype of cloche mutant may be a novel point mutation allele of the cloche mutant.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis. METHODS: In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80. CONCLUSION: TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.

[Expression of oxyntomodulin in bifidobacteria and effect of oxyntomodulin-transformed bifidobacteria on the body weight of obese mice].

OBJECTIVE: To observe the effect of pBBADs-OXM-transformed bifidobacteria on the body weight of obese mice. METHODS: B. longum was transformed with pBBADs-OXM by electroporation, and arabopyranose-induced oxyntomodulin expression by the bacterium was detected by ELISA. pBBADs-OXM-transformed bifidobacteria was administered orally obese mice on a daily basis with pBBADs-GFP-transformed bifidobacteria as the negative control, and the body weight changes of the mice were observed. RESULTS: OXM was detected by ELISA not only in the supernatant but also the precipitant of the transformed bacterial culture. The body weight of the obese mice fed with pBBADs-OXM-transformed bifidobacteria decreased significantly compared with that of the mice in the obese model group (P<0.05). CONCLUSION: Administration of pBBADs-OXM-transformed B.longum can reduce the body weight of obese mice.

Hydrogen peroxide induces G2 cell cycle arrest and inhibits cell proliferation in osteoblasts.

Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5-200 microM hydrogen peroxide (H(2)O(2)) dose- and time-dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3-E1, and in human osteoblast-like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G(2)/M phase increased about 2-4 fold by 200 microM H(2)O(2) treatment for 24-72 hr. A total of 0.5-5 mM of H(2)O(2) but not lower concentrations (5-200 microM) of H(2)O(2) inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and S6(S235/236) in MC3T3-E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H(2)O(2) prevents cell proliferation of osteoblasts by down-regulating cyclin B1 and inducing G(2) cell cycle arrest. Inhibition of mTOR signaling by H(2)O(2) may not be involved in this process.

[Role of phospholipase C-gamma1 signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells].

OBJECTIVE: To explore the role of phospholipase C-gamma1 (PLC-gamma1) signaling pathway in H(2)O(2)-induced apoptosis of PC12 cells. METHODS: PC12 cells were exposed to 50 micromol/L H(2)O(2) after pretreatment with 10 micromol/L U73122, a specific PLC-gamma1 inhibitor. Hoechst/PI double staining was performed to observe the morphological changes of the cells under light microscope. MTT assay was used to evaluate the cell viability, and the percentage of apoptotic cells was analyzed by flow cytometry. DNA fragmentation assay was carried out to characterize the cell apoptosis. RESULTS: After inhibition of the PLC-gamma1 signaling pathway with 10 micromol/L U73122, PC12 cells showed obvious apoptotic morphology, the viable cells decreased significantly, and the percentage of apoptotic cells rose to 35.7%. PC12 cells treated with U73122 presented with a distinct DNA ladder on electrophoresis resulting from DNA cleavage in the apoptotic cells. CONCLUSION: PLC-gamma1 signaling pathway plays an important protective role in H(2)O(2)-induced PC12 cell apoptosis.

[BRCA1 regulates progesterone receptors A and B protein expressions in breast cancer cells in vitro].

OBJECTIVE: To study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells. METHODS: Breast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting. RESULTS: The protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased. CONCLUSION: In breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.

[Construction and expression of different mutants of human p53 and their effects on arsenite-induced cell apoptosis].

OBJECTIVE: To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis. METHODS: Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry. RESULTS: The recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells. CONCLUSION: The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.

[Matrine-induced erythroid differentiation of K562 cells is associated with activation of the apoptotic pathway].

OBJECTIVE: To observe matrine-induced erythroid differentiation of K562 cells in relation to activation of the apoptotic pathway in vitro. METHODS: K562 cells were cultured in the presence or absence of matrine at different concentrations for 4 days, and the morphological and ultramicrostructural changes of the cells were observed using inverted microscopy and transmission electron microscopy, respectively. The expression of apoptosis-related protein p27kip1 was detected by immunocytochemistry. RESULTS: Compared to untreated K562 cells, the cells treated with matrine at 0.10 g/L exhibited apoptostic characteristics in the cellular morphology and ultramicrostructure, with the expression of p27kip1 protein upregulated in a concentration- and time-dependent manner. CONCLUSION: Matrine-induced erythroid differentiation of K562 cells is associated with cell apoptosis, and upregulation of p27kip1 protein expression may play a crucial role in the process of apoptosis.

[Prokaryotic expression and bioactivity of human platelet-derived growth factor B chain mature peptide].

OBJECTIVE: To express human platelet-derived growth factor (hPDGF) B chain mature peptide gene in a prokaryotic expression system and detect the bioactivity of the expressed protein. METHODS: hPDGF B chain mature peptide gene was amplified and expressed in E. coli, and the recombinant protein, rhPDGF-BB, was purified and renatured in GSSG/GSS system. The bioactivity of rhPDGF-BB in vitro was evaluated with SD rat osteoblasts. RESULTS: The full-length PDGF-B mature peptide gene was obtained and verified, and successfully expressed in E. coli. Bioactivity detection results showed that the expressed rhPDGF-BB obviously promoted the proliferation and DNA replication of SD rat osteoblasts in vitro (P<0.01). CONCLUSION: he PDGF-B chain mature peptide cDNA has been successfully cloned and the PDGF-B precursor highly expressed in E. coli, and renatured rhPDGF-BB displays high bioactivity as shown by MTT assay and flow cytometry. This success provides the basis for production of functional PDGF-BB and facilitates further studies of its role in fracture healing and trauma reconstruction.

[Effects of blocking phospholipase C-gamma1 signaling pathway on proliferation and apoptosis of human colorectal cancer cell line LoVo].

BACKGROUND & OBJECTIVE: Phospholipase C-gamma 1 (PLC-gamma1) is a vital signal transducer in transmembrane signaling, which regulates cell proliferation and apoptosis. It is overexpressed in many cancers, such as colorectal cancer, which indicates that it is closely related to the genesis and development of tumors. This study was to explore the effects of blocking PLC-gamma1 signaling pathway on the proliferation and apoptosis of human colorectal cancer cell line LoVo, and investigate the signaling mechanisms. METHODS: LoVo cells were treated with PLC-gamma1-specific chemical blocking agent U73122. Cell proliferation was examined by cell counting, MTT assay, and flow cytometry (FCM). Cell apoptosis was observed under a microscope, and measured by agarose gel electrophoresis and FCM with PI simple staining. The expression of hot shock protein 70(HSP70) and Caspase-3 in LoVo cells were detected by Western blot. RESULTS: The proliferation of LoVo cells was inhibited after blocking PLC-gamma1 signaling pathway and the effect was enhanced along with the increasing concentration of U73122. The inhibition rate reached 35% and 45% when treated with 10 micromol/L U73122 for 24 h and 48 h respectively. After blocking PLC-gamma1 signaling pathway, the G1 phase proportion of LoVo cells was increased while the S phase proportion was decreasedû no apoptosis-specific cell shrinkage was found under a light microscope, and no apoptosis-specific DNA ladder was found by agarose gel electrophoresisû no activated Caspase-3 was detected by Western blot, while increased expression of HSP70 was detected. CONCLUSIONS: Blocking PLC-gamma1 signaling pathway can inhibit the proliferation and cell cycle progress of LoVo cells, which may be due to the up-regulated expression of HSP70. PLC-gamma1 is not a vital signal molecule regulating the apoptosis of LoVo cells.

[Adenovirus-mediated overexpression of AMP-activated protein kinase induces LX2 cell apoptosis].

OBJECTIVE: To observe the effect of adenovirus-mediated overexpression of AMP-activated protein kinase (AMPK) on apoptosis of hepatic stellate cell line LX2. METHODS: After adenovirus infection of the LX2 cells, the exogenous gene expression was detected by Western blotting, and cell apoptosis evaluated by flow cytometry with PI staining. Agarose gel electrophoresis was performed to detect the DNA ladder, and the expressions of caspase-3, Bcl-2 and Bax are detected by Western blotting. RESULT: With AMPK overexpression, apoptotic peak and DNA ladder of the infected cells appeared, and pro-caspase-3 was activated to transform into caspase-3 accompanied by up-regulated Bax expression, whereas Bcl-2 expression was hardly detectable by Western blotting. CONCLUSION: Overexpression of AMPK mediated by the adenovirus can induce LX2 cell apoptosis, possible as a result of up-regulated Bax expression with low Bcl-2 expression.