Animals ; Gene Expression Regulation ; drug effects ; Gene Targeting ; methods ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAbeta) is required to induce typical symptoms in host plants. DNAbeta encodes a single gene (termed betaC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing the betaC1 gene of a DNAbeta associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing betaC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the betaC1 protein showed that the betaC1 protein accumulated in the nuclei of cells. The possible biological function of the betaC1 protein was discussed.
Cell Division ; physiology ; Cell Nucleus ; genetics ; metabolism ; ultrastructure ; Cells, Cultured ; DNA, Viral ; genetics ; Geminiviridae ; genetics ; Plant Diseases ; genetics ; virology ; Plant Leaves ; cytology ; genetics ; growth & development ; metabolism ; Plants, Genetically Modified ; growth & development ; metabolism ; Recombinant Proteins ; metabolism ; Tobacco ; cytology ; growth & development ; metabolism ; ultrastructure ; Viral Proteins ; genetics ; metabolism

Background There are a lot of studies about the carrier of corneal endothelial transplantation,but the best carrier has not been defined.Objective This study was to investigate the biocompatibility of chitosan carrier with rabbit corneal endothelium in vivo.Methods Fresh eye-balls were obtained from 10 New Zealand white rabbits.Rabbit corneal endothelial cells (CECs) were isolated and cultured on chitosan carrier in vitro.The morphology and density of rabbits CECs were observed every day,and the expressions of fibronectin (FN),collagen-1 (Coil-I) and Zonula occludens 1 (ZO-1) were detected by immunoinfluorescence.The morphology and ultrastructure of CECs were observed under the scanning and transmission electron microscope.Chitosan carrier with CECs was implanted into the anterior chamber of the left eyes in ten healthy New Zealand white rabbits,and only paracentesis of anterior chamber was performed in the right eyes as controls.The inflammation of ocular anterior segment was examined under the slit lamp microscope,and corneal thickness was measured 1 week,4 and 8 weeks after operation.Corneal endothelium cell density and morphology were examined under the corneal endothelial microscope at postoperative 2 weeks.Corneal samples were collected for the regular histopathological examination to observe the inflammatory reaction at postoperative 1 month and 3 months.Paired t test was used for statistical analyses between the control group (left eyes) and the experimental group (right eyes).The use and care of the animals followed the Statement of ARVO.Results CECs formed an intact monolayer of cells with the uniform shape and size on the chitosan membrane after incubated for 5 days.The cells reached confluence of 90％ 7 days after cultured with the 40％ hexagon cells.Under the scanning electron nicroscope,rabbit CECs showed the round or polygon in the shape with the microvillus on the cell surface.The cells connected closely by desmosome.The processes,pseudopodiums and microvillus on the cellular surface,vacuole in the cytoplasm,expanded endoplasmic reticulum with ribosome and abundant chromatin were exhibited under the transmission electron microscope.The immunofluorescence examination revealed the positive expressions of FN,Coll-Ⅰ and ZO-1 in the CECs on the chitosan carrier.In the in vivo experiment,the exudation in the anterior chamber and corneal edema were seen under the slit lamp microscope 3 days after implantation of chitosan carrier with CECs.However,the inflammation was gone 14 days after operation.The differences of the corneal thickness were no significant between the experimental group and the control group 1 week and 4,8 weeks after operation (t =1.377,P=0.265;t =1.795,P=0.165 ; t =0.390,P =0.760).In addition,no significant differences were found in the CECs density and the hexagon cells rate between the two groups(P =0.365,0.062).The histopathological examination showed that the inflammatory cells around the chitosan membrane were disappeared 3 months after operation and showed a good corneal structure.Conclusions Chitosan carrier has a good biocompatibility with rabbit CECs and anterior chamber,and it may be a potentially good carrier for CECs transplantation.

Bacterial biodiversities of microbial mat and sediments, which were sampled from thermal springs of Tengchong Rehai in Yunnan, were preliminarily studied with PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Directly extracted total DNA from environmental samples amplified by PCR with two sets of bacteria-specific primers. The PCR products, which include the V 8 and V 9 high-variable regions respectively, were analyzed by using DGGE. The DGGE profiles not only indicated the existence of higher levels of bacterial diversity, but also showed that the microbial mat and sediments have different dominant bacteria. Furthermore, the bacterial PCR-DGGE displayed clear profiles of bacterial structure selected by the key abiotic factors of the extreme environments, such as temperature and concentration of oxygen.

COP9 Signalosome Complex ; Carcinoma, Hepatocellular ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; chemistry ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Neoplastic ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Peptide Hydrolases ; chemistry ; genetics ; metabolism ; Phosphorylation ; Phosphoserine ; metabolism

BACKGROUND:Gemcitabine hydrochloride is a water-soluble anticancer drug that induces apoptosis in tumor cells, but it has an excessive release in vivo. OBJECTIVE:To evaluate the effect of poly(lactic acid) (PLA) electrospun fiber membranes carrying gemcitabine hydrochloride on the growth of human osteosarcoma cell lines MG-63. METHODS:PLA electrospun fiber members with (experimental) or without (control) gemcitabine hydrochloride were fabricated and characterized. Two kinds of fiber membranes were immersed in low-glucose DMEM medium, and the supernatants were collected in the two groups at 3, 5, 7 days, respectively. Passage 5 human osteosarcoma cell lines MG-63 were inoculated into 96-well plates containing low-glucose DEME with 15％ fetal bovine serum, and divided into seven groups. Groups 1-3 were cultured in the experimental supernatants of 3, 5, 7 culture days, and groups 4-6 were cultured in the control supernatants of 3, 5, 7 culture days, respectively. The remaining group acted as the negative control with no supernatant. Thereafter, cell counting kit-8 was used to detect cell proliferation, and RT-PCR was used to measure expression of Bcl-2 and Bax at 3 days of culture. RESULTS AND CONCLUSION:(1) No obvious particle was found on the smooth and even surface of the fiber members in the experimental and control groups. There was no significant difference in fiber diameter, contact angle and tensile strength between the two kinds of fiber membranes. (2) The results of cell counting kit-8 showed that compared with the negative control group, the supernatant released from the control group had no effect on the MG-63 proliferation at different time points, while the supernatant released from the experimental group could inhibit the MG-63 proliferation at different time points (P<0.05), and the inhibitory effect became more and more obvious with the prolongation of release time. (3) RT-PCR findings showed that compared with the control group, the supernatant released from the experimental group could increase Bax mRNA expression and decrease Bcl-2 mRNA expression at the same time point. To conclude, the PLA electrospun fiber membranes carrying gemcitabine hydrochloride can sustainably inhibit MG-63 proliferation and promote cell apoptosis.

To investigate changes of 14-3-3beta from apoptosis induced by PrP106-126 polypeptide, HeLa cell was incubated with PrP106-126 for 4h or 8h. Nucleus changes and the expression of PARP were detected differently by Hoechst staining and Western blotting. Expressing of protein and mRNA from 14-3-3beta was determined by Western blotting and Real-time PCR. The results show that typical nucleus pyknosis and chip of apoptosis and degradation of PARP were induced by PrP106-126 peptide in HeLa cells. Degradation of 14-3-3beta appeared in apoptosis groups induced by PrP106-126 peptide. However, 14-3-3beta mRNA did not display any changes in apoptosis groups. This study indicated that degradation of antiapoptosis protein 143-3beta induced by PrP106-126 peptide may be one of pathogenesis mechanism of prion disease.
14-3-3 Proteins ; metabolism ; Apoptosis ; drug effects ; HeLa Cells ; Humans ; Peptide Fragments ; pharmacology ; Prions ; pharmacology ; Proteolysis ; drug effects

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

UNLABELLEDTo investigate the relationship between Ureaplasma urealyticum (UU) infection and semen quality.

METHODSFrom 2001 to 2003, 346 eligible patients aged 20-45 years were invited from two hospitals in Shanghai, China, to participate in an investigation which included questionnaires about general and reproductive health, an external genital tract examination, UU culture and semen analysis. Multiple linear regression models were used to examine whether UU had a significant effect on semen quality after adjustment for confounding factors.

RESULTSFindings suggested that UU infection was associated with higher semen viscosity and lower semen pH value. Sperm concentration was lower in UU positive subjects than that in UU negative subjects (54.04 X 10(6)/mL vs.70.58 X 10(6)/mL). However, UU did not significantly affect other semen quality indexes.

CONCLUSIONUU infection of the male genital tract could negatively influence semen quality.

Adult ; Clothing ; Humans ; Infertility, Male ; epidemiology ; Male ; Middle Aged ; Physical Examination ; Semen ; physiology ; Sperm Count ; Sperm Motility ; Spermatozoa ; physiology ; Surveys and Questionnaires ; Testis ; anatomy & histology ; Ureaplasma Infections ; physiopathology ; Ureaplasma urealyticum

OBJECTIVETo evaluate the efficacy and toxicity of taxol-based chemotherapy in the treatment of advanced gastric cancer (AGC).

METHODSTwenty-nine patients with AGC treated with taxol-based protocols: taxol plus 5-fluouracil 17 patients, taxol plus cisplatin 10 patients, taxol plus epirubicin 2 patients.

RESULTSTwenty-six patients were evaluated for clinical response. There was no complete response, PR 10 (34.5%), SD 12 (41.4%), PD 4 (13.8%). The total response rate was 34.5%. The clinical beneficial response rate was 72.4% (21/29). Median time to progression (mTTP) was 5.8 months and median overall survival time was 9.3 months. The main side effects were: suppression of bone marrow in 26 patients (89.7%), alopecia in 25 (86.2%), myodynia and arthrodynia in 23 (79.3%). There was no death during the treatment.

CONCLUSIONTaxol-based chemotherapy is an effective and well tolerated regimen in the treatment of AUC, which can relieve suffering and improve quality of life of the patients. It can be used as the second-line therapy for relapsed advanced gastric cancer.

Adult ; Aged ; Alopecia ; chemically induced ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cisplatin ; administration & dosage ; Drug Administration Schedule ; Epirubicin ; administration & dosage ; Female ; Fluorouracil ; administration & dosage ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Paclitaxel ; administration & dosage ; adverse effects ; Quality of Life ; Remission Induction ; Stomach Neoplasms ; drug therapy ; pathology