The purpose was to compare the difference between transgene expressions driven by homologous duplicated carbonic anhydrase (DCA) promoter and foreign CaMV35S promoter in the unicellular green alga, Dunaliella salina(D.salina).The CaMV35S promoter-bar construct and DCA promoter-bar construct into D.salina by a Backon 2000 electroporation system were introduced. After the repeated selections with the phosphinothricin (PPT) of 3mg/L, 3 PPT-resistant phenotype transformants were isolated from the CaMV-bar and DCA-bar pools of transformants of D. salina, respectively. The results of PCR and sequencing showed that bar genes were stably integrated into the genome of D.salina, and Southern bolts showed the number of transgene copy had no significant difference between both promoters. Semi-quantitive RT-PCR indicated that the mRNA levels of bar gene were higher in DCA-bar transformants than the CaMV-bar transformants, and could be increased under the induction of high salt in DCA-bar transformants but not in the CaMV-bar transformants. Analysis of growth rate of transformants showed DCA-bar transformants achieved the log stage faster than the CaMV-bar transformants. It is concluded that the homologous promoters have more advantages than the foreign promoters in the transgenic D.salina.

BACKGROUND: In valerian-ligusticum extract (VLE), valeriana offici nalis extract (VOE) is γ aminobutyric acid (GABA) receptor kinetin, which can relax cerebral vascular spasm; ligusticum wallichii Fr. Extraxt (LWE)can pass through blood-brain barrier, enhance microcirculation of tissue and inhibit blood platelet aggregation and 5-Hydroxytryptamine (5-HT) release.OBJECTIVE: To probe into the effects of VLE prepared with effective components on prevention and treatment of cerebral ischemic injury.DESIGN:Complete randomized, negative and positive control experiment.SETTING: Institute of Senile Medicine and Pharmacology of Tongji Medical College of Huazhong University of Science and Technology.MATERIALS: The experiment was performed in Institute of Senile Medicine Pharmacology of Tongji Medical College of Huazhong University of ent blood perfusion in brain tissue: Fifty Kunming mice were employed,which was randomized into normal group, solvent control (model) group,ligustrazine 50 mg/kg group, VLE 170 mg/kg group and VLE 85 mg/kg Fifty Wistar rats were employed, which was randomized into solvent control (model) group, compound danshen (Radix Salviae Miltiorrhizae) 5 g/kg group,VLE 156 mg/kg group, VLE 94 mg/kg group and VLE 31.3 mg/kg group,Sixty Wistar rats were employed, which was randomized into sham-operation group, solvent control (model) group, ligustrazine 10 mg/kg group, VLE 156 mg/kg group, VLE 95 mg/kg group and VLE 31.3 mg/kg, 10 mice in each were employed, which was randomized into normal group, solvent control (model) group, ligustrazine 10 mg/kg group, VLE 200 mg/kg group and VLE 40 mg/kg, 10 mice in each one.sue, in advance, VLE (85, 170 mg/kg), ligustrazine (50 mg/kg) or solvent enhancer of equal volume (0.2 mL) were injected abdominally in each group. Twenty minutes later, pituitrin (2.5 u/kg) was injected intravenously; and 10 minutes later, isotope 99Tcm+ L, L-EthylCysteinate Dimer and Stannous Chloride (ECD) 3.7×1010Bq/ L(0.1 mL/per mouse) was injected in coccygeal nerve. Fifteen minutes later, radio-immunity counter was used periment of arteral-ovenous bypass method for thrombosis, before the opercal saline successively, continuously for 7 days, once per day. After 24 hours of medication pause, with abdominal anesthesia with pentobarbitol sodium, a catheter (with surgical thread inside) was used in vitro to connect common cervical vein and carotid artery. Thrombus mass was scaled 15 dominal anesthesia of chloral hydrate, intraluminal thread approach (ITA)was used to block unilateral MCA. Except that ITA was not used, the other management in sham-operation group was same as experimental groups.Gastric perfusion was done with VLE(156, 94, 31.3 mg/kg), ligustrazine operation and 3 hours and 12 hours after operation. 24 hours after modeling, the assessment was done for behavioral neurological damage and brain sive cerebral ischemia experiment, the model was prepared by coccygeal injection of collagen + adrenalin (AD). Respectively, 30 minutes before modeling injection and 1 hour after injection, gastric perfusion was done with VLE (200, 40 mg/kg), ligustrazine (10 mg/kg) or solvent enhancer of equal volume successively to observe the numbers of dead mice in 5 minutes after modeling and the numbers of hemiplegia mice in 15 minutes;and to determine brain mass index 8 hours later after sacrificed and lactic acid level of brain tissue homogenate with ultraviolet spectrophotometry.group.RESULTS: In the experiment of acute extensive brain ischemia in mice, in solvent control, during modeling, 3 mice were died and the rest 207 mice brain tissue in mice, the ratios of brain with and blood γ ray pulsating intensity in VLE 85 mg/kg group and VLE 170 mg/kg were higher than model group (0.53±0.09, 0.55±0.08, 0.45±0.08, t=2.234 6, 2.793 3, P method in rats, the thrombus masses in VLE 156 mg/kg group, 94 mg/kg group and 31.3 g/kg group were lower remarkably than the model group [(12.66±4.79), (13.31 ±3.97), (13.49±4.09), (19.21±5.76) g, (t=2.667 0,31.3 mg/kg group, 94 mg/kg group and 156 mg/kg group was lower remarkably than model group successively [(5.9±1.9), (6.0±2.0), (5.8±2.2),(8.7±0.9) score], and cerebral infarction index was lower than model group [(16.52±5.78)%,(16.54±3.00)%, (14.18±6.13)%, (24.03±4.85)%, (t=3.118 9-chemia in mice, brain mass indexes of VLE 40 mg/kg and 200 mg/kg groups were lower remarkably than model group [(0.91 ±0.20) and (0.82±0.24)%, (1.40±0.32)%], and lactic acid in brain tissue was lower than model group [(17.44±6.71),(14.43±2.81), (29.07±7.33) μmol/g (t=3.388 5-5.800 5, P＜ 0.01)].CONCLUSION: Valerian-liqusticum extract improves significantly cerebral ischemia in mice induced by pituitrin and the damage by medium cerebral artery embolism in rats, and it inhibits significantly blood platelet aggregation and thrombosis induced by AD+ collagen mixture or foreign objects. It is suggested that valerian-ligustrazine extract prevents and treats significantly the perfusion disturbance of cerebral microcirculation.

Objective To investigate the radiosensitivity effects of poly ADP-ribose polymerase-1 (PARP-1) inhibitor 3-amion benzamide (3-AB) on the BRCA non-mutant and BRCA mutant breast cancer cells,and to explore the regulatory mechanism of PARP-1 and BRCA in radiation-induced DNA damage repair.Methods MDA-MB-436 cells and MDA-MB-231 cells were divided into four groups respectively as the control (CTRL),ionizing radiation alone (IR),3-AB alone (3-AB),and ionizing radiation combined with 3-AB(IR + 3-AB)group.γ-H2AX foci were detected by immunofluorescence assay.The radiosensitivity of breast cancer cells was evaluated by clonogenic survival assay.The percentage of apoptotic cells was assessed by flow cytometry.Results Compared with MDA-MB-231 cells,MDA-MB-436 cells had a higher radiosensitivity and produced more γ-H2AX foci(t =4.57,P ＜ 0.05),which was further increased by 3-AB.The DNA damage of MDA-MB-436 cells in the IR + 3-AB group was the most remarkable (t =3.26,P ＜ 0.05).Flow cytometry showed that the cells in the IR + 3-AB group had the highest rate of apoptosis (t=3.81,P ＜ 0.05),and the apoptosis rate of MDA-MB-436 cells was significantly higher than MDA-MB-231 cells (t =2.96,P ＜ 0.05).Conclusions The radiosensetivity of BRCA mutant cells MDA-MB-436 is significantly higher than that of non-BRCA mutant cells MDA-MB-231.Inhibition of PARP-1 can further increase the apoptosis and radiosensitivity of BRCA-mutant cells by further blocking the repair of DNA single-strand break induced by ionizing radiation.

This study assessed the effects of leukemia-related protein 16 (LRP16) on the regulation of pancreatic functions in mouse insulinoma (MIN6) cells. Cells with down-regulated expression of LRP16 were obtained by a shRNA interference strategy. Insulin content and glucose-stimulated insulin secretion (GSIS) were examined by radioimmunoassay. Western blotting was applied to detect protein expression. Glucose-stimulated sub-cellular localization of PDX-1 was immunocytochemically determined. Cell proliferation and apoptosis were detected by flow cytometry. Our results showed that LRP16 regulated insulin content in MIN6 cells by controlling expression of insulin and insulin transcription factors. LRP16 gene silence in MIN6 cells led to reduced cell proliferation and increased apoptosis. The observation of phosphorylation of serine-473 Akt and the localization of PDX-1 to the nucleus under glucose-stimulation exhibited that LRP16 was a component mediating Akt signaling in MIN6 cells. These results suggest that LRP16 plays a key role in maintaining pancreatic β-cell functions and may help us to understand the protective effects of estrogen on the functions of pancreatic β-cells.

Clinical characteristics,including primary and secondary sexual characteristics,basal endocrine profiles,and imaging results were reviewed.Follow-up data were recorded.16 patients had normal karyotypes,manifest amastia,infantile genitalia,amenorrhea,and delayed epiphyseal fusion at the knee and wrist joints.Serum gonadotropic hormone levels were significantly below normal values.15 patients underwent a gonadotropin-releasing hormone (GnRH) stimulation test and 6 patients had a prolonged GnRH stimulation test.16 patients underwent pituitary or brain magnetic resonance imaging (MRI),which showed small pituitaries in three patients,wing tips of suspicious nodules in 2 patients,an empty sella turcica in 1 patient,and a missing right olfactory bulb and tract in 1 patient.1 patient had no detectable uterus or accessory organs,while the other patients had primordial uteri.1 patient was diagnosed as a case of severe osteoporosis.1 patient suffered from pituitary stalk interruption syndrome.An artificial menstrual cycle due to hormone replacement therapy was not sustained after discontinuation of hormone therapy.As disease severity and the date of initiating hormone replacement varied,the results of treatment were quite different.For patients of reproductive age,it was rare to see a reversal of idiopathic hypogonadotropic hypogonadism after discontinuation of hormone therapy.

BACKGROUNDMutations in fumarylacetoacetate hydrolase (FAH) gene can lead to tyrosinemia type 1 (HT1), a relatively rare autosomal recessive disorder. To date, no molecular genetic defects of HT1 in China have been described. We investigated a Chinese family with a HT1 child to identify mutations in FAH.

METHODSDNA sequencing was used for mutations screening in FAH gene. Real-time polymerase chain reaction (PCR) was performed to determine the FAH gene expression level. To confirm the presence of degradation by the nonsense-mediated mRNA decay pathway (NMD), the fragments containing R237X mutations were analyzed by primer introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and cDNA sequencing. Finally, the effects of the mutations reported in this study were predicted by online softwares.

RESULTSA boy aged 3 years and 8 months was diagnosed clinically with HT1 based on his manifestations and biochemical abnormalities. Screening of FAH gene revealed two heterozygous mutations R237X and L375P transmitted from his mother and father respectively. In this pedigree, the amount of FAH mRNA relative to a healthy control was 0.44 for the patient, 0.77 for his mother and 1.07 for his father. Moreover, both PIRA-PCR and cDNA sequencing showed significant reduction of the FAH mRNA with R237X nonsense mutation. The missense mutation of L375P was not reported previously and prediction software showed that this mutation decreased the stability of protein structure and affected protein function.

CONCLUSIONSThis is the first case of HT1 analyzed by molecular genetics in China. The R237X mutation in FAH down- regulates the FAH gene expression, and the L375P mutation perhaps interrupts the secondary structure of FAH protein.

Child, Preschool ; China ; Humans ; Hydrolases ; genetics ; Male ; Molecular Sequence Data ; Mutation ; Mutation, Missense ; genetics ; Nonsense Mediated mRNA Decay ; genetics ; Real-Time Polymerase Chain Reaction ; Tyrosinemias ; genetics

Laser-induced breakdown spectroscopy (LIBS) was used for analysis of the distribution of S,Mn,Fe,Cr,Mo,Si,Al in a 34CrNiMo6 steel sample cut from a main shaft of wind driven generator.The MnS inclusion area in each ablation craters cover zone was extracted in the way of comparing the metallograph captured by optical microscopy before and after LIBS scanning ablation.The statistic relation between MnS inclusion area and signal intensity of S and Mn was analyzed.The result showed that the abnormal signal of S and Mn occurred at the same position with the existence of MnS inclusion,and their signal intensity showed linear relationship.The abnormal signal of S and Mn were triggered mainly by MnS inclusion.The statistic result also showed linear relationship between signal intensity and MnS inclusion area both for S and Mn.It was possible to determine the inclusion type,size and distribution by analyzing abnormal signal.A simplified ablation model was established to calculate the relation of S and Mn content to MnS inclusion area.The arithmetic result showed a linear relation between the content and MnS inclusion area both for S and Mn.The calculation confirmed the linear relationship between signal intensity and inclusion area observed in experiment.The linear relationship could be interfered by macro-segregation,micro-segregation,deviation in measuring inclusion area,and inclusion spatter in pre-ablation.

BACKGROUND: The present study aimed to determine the short-term and long-term outcomes of critically ill patients with acute respiratory insufficiency who had received sedation or no sedation. METHODS: The data of 91 patients who had received mechanical ventilation in the first 24 hours between November 2008 and October 2009 were retrospectively analyzed. These patients were divided into two groups: a sedation group (n=28) and a non-sedation group (n=63). The patients were also grouped in two groups: deep sedation group and daily interruption and /or light sedation group. RESULTS: Overall, the 91 patients who had received ventilation ≥48 hours were analyzed. Multivariate analysis demonstrated two independent risk factors for in-hospital death: sequential organ failure assessment score (P=0.019, RR 1.355, 95％CI 1.051–1.747, B=0.304, SE=0.130, Wald=50483) and sedation (P=0.041, RR 5.015, 95％CI 1.072–23.459, B=1.612, SE=0.787, Wald=4.195). Compared with the patients who had received no sedation, those who had received sedation had a longer duration of ventilation, a longer stay in intensive care unit and hospital, and an increased in-hospital mortality rate. The Kaplan-Meier method showed that patients who had received sedation had a lower 60-month survival rate than those who had received no sedation (76.7％ vs. 88.9％, Log-rank test=3.630, P=0.057). Compared with the patients who had received deep sedation, those who had received daily interruption or light sedation showed a decreased in-hospital mortality rate (57.1％ vs. 9.5％, P=0.008). The 60-month survival of the patients who had received deep sedation was significantly lower than that of those who had daily interruption or light sedation (38.1％vs. 90.5％, Log-rank test=6.783, P=0.009). CONCLUSIONS: Sedation was associated with in-hospital death. The patients who had received sedation had a longer duration of ventilation, a longer stay in intensive care unit and in hospital, and an increased in-hospital mortality rate compared with the patients who did not receive sedation. Compared with daily interruption or light sedation, deep sedation increased the in-hospital mortality and decreased the 60-month survival for patients who had received sedation.

This study assessed the effects of leukemia-related protein 16 (LRP16) on the regulation of pancreatic functions in mouse insulinoma (MIN6) cells.Cells with down-regulated expression of LRP16 were obtained by a shRNA interference strategy.Insulin content and glucose-stimulated insulin secretion (GSIS) were examined by radioimmunoassay.Western blotting was applied to detect protein expression.Glucose-stimulated sub-cellular localization of PDX-1 was immunocytochemically determined.Cell proliferation and apoptosis were detected by flow cytometry.Our results showed that LRP16 regulated insulin content in MIN6 cells by controlling expression of insulin and insulin transcription factors.LRP16 gene silence in MIN6 cells led to reduced cell proliferation and increased apoptosis.The observation of phosphorylation of serine-473 Akt and the localization of PDX-1 to the nucleus under glucose-stimulation exhibited that LRP16 was a component mediating Akt signaling in MIN6 cells.These results suggest that LRP16 plays a key role in maintaining pancreatic β-cell functions and may help us to understand the protective effects of estrogen on the functions of pancreatic β-cells.

OBJECTIVETo investigate the value of intensity modulated radiation therapy (IMRT) for patient with gynecological malignancies after treatment of hysterectomy and chemotherapy/radiotherapy.

METHODSAll 32 patients with cervical or endometrial cancer after hysterectomy received full course IMRT after 1 to 3 cycles of chemotherapy (Karnofsky performance status(KPS) > or =70). Seventeen of these patients underwent postoperative preventive irradiation and the other 15 patients were pelvic wall recurrence and/or retroperitoneal lymph node metastasis, though postoperative radiotherapy and/or chemotherapy had been given after operation.

RESULTSThe median dose delivered to the PTV was 56.8 Gy for preventive irradiation, and 60.6 Gy for pelvic wall recurrence or retroperioneal lymph node metastasis irradiation. It was required that 90% of iso-dose curve could covere more than 99% of GTV. However, The mean dose irradiated to small intestine, bladder, rectum, kidney and spinal cord was 21.3 Gy, 37.8 Gy, 35.3 Gy, 8.5 Gy, 22.1 Gy, respectively. Fourteen patients presented grade I (11 patients) or II (3 patients) digestive tract side-effects, Five patients developed grade I or II bone marrow depression. Twelve patients had grade I skin reaction. The overall 1-year survival rate was 100%. The 2- and 3- year survival rate for preventive irradiation were both 100%, but which was 5/7 and 3/6 for the patients with pelvic wall recurrence or retroperioneal lymph node metastasis.

CONCLUSIONIntensity modulated radiation therapy can provide a better dose distribution than traditional radiotherapy for both prevention and pelvic wall recurrence or retroperioneal lymph node metastasis. The toxicity is tolerable. The adjacent organs at risk can well be protected.

Adult ; Aged ; Combined Modality Therapy ; Diarrhea ; etiology ; Endometrial Neoplasms ; drug therapy ; radiotherapy ; surgery ; Female ; Follow-Up Studies ; Humans ; Hysterectomy ; methods ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Recurrence, Local ; Radiotherapy Dosage ; Radiotherapy, Intensity-Modulated ; adverse effects ; methods ; Survival Analysis ; Uterine Cervical Neoplasms ; drug therapy ; radiotherapy ; surgery