In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.
Genetic Markers ; Green Fluorescent Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Plasmids ; Recombination, Genetic ; Tobacco ; genetics

AIMTo study the effects of 1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino) propane hydrochloride(DDPH) on brain ischemia injury in rats.

METHODSBy using the middle cerebral artery occlusion (MCAO) induced by nylon surgical thread inserted through the internal carotid artery into the anterior cerebral artery in rats, the effects of DDPH on neuron defects(ND) and infarct size(IS) were investigated. Using incomplete cerebral ischemia in rats, the effects of DDPH on superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain tissue and pathological changes in rats were studied.

RESULTSDDPH at the dose of 10 mg.kg-1 i.p. 30 min before ischemia decreased the ND 3 h after ischemia. The IS declined 24 h after ischemia as well. Meanwhile, DDPH was found to increase SOD activity and reduce the MDA content, as well as mitigate pathological damage, of neuron after brain ischemia in rats.

CONCLUSIONDDPH showed protective effects on brain ischemia, probably related to its properties of calcium antagonistic effect and increasing the activity of superoxide dismutases.

Animals ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; Female ; Infarction, Middle Cerebral Artery ; pathology ; Male ; Malondialdehyde ; metabolism ; Neuroprotective Agents ; pharmacology ; Phenethylamines ; pharmacology ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; pathology ; Superoxide Dismutase ; metabolism

BACKGROUNDTanis was reported as a putative receptor for serum amyloid A (SAA) involving glucose regulated protein in insulin regulated resistance. It was found to be dysregulated in diabetic rats (Psammomys obesus, Israeli sand rat) and its homologue for humans is SelS/AD-015. The present study analyzed mRNA expression of SelS in omental adipose tissue biopsies from patients with type 2 diabetes mellitus (T2DM), and age- and weight-matched nondiabetic patients, the relationship of SelS mRNA with Homa-IR and serum SAA level.

METHODSHuman omental adipose tissues from ten cases of type 2 diabetic patients and twelve cases of nondiabetic individuals were analyzed for the expression level of SelS mRNA by semiquantitative polymerase chain reaction (PCR), Homa-IR estimated by standard formula and SAA level by enzyme-linked immunosorbent assay (ELISA).

RESULTSSelS mRNA expression, Homa-IR and serum SAA were higher in T2DM sufferers than in nondiabetic control group. SelS mRNA level was positively correlated with Homa-IR and SAA level in each group.

CONCLUSIONSSelS protein may be involved in insulin resistance in Chinese with T2DM by acting as the SAA receptor, thus playing an important role in the development of T2DM and atherosclerosis.

Adipose Tissue ; metabolism ; Adult ; Aged ; Base Sequence ; Diabetes Mellitus, Type 2 ; metabolism ; Female ; Humans ; Insulin Resistance ; Male ; Membrane Proteins ; genetics ; Molecular Sequence Data ; Omentum ; metabolism ; RNA, Messenger ; analysis ; Selenoproteins ; genetics ; Serum Amyloid A Protein ; analysis

BACKGROUNDA novel anti-rheumatic drug, T-614, has been shown to have an anti-inflammatory effect and to improve abnormal immunological findings in rheumatoid arthritis (RA). To assess the safety and efficacy of T-614 versus placebo in patients with active RA we conducted a 24-week clinical study in 280 Chinese patients.

METHODSIn a multicenter, randomized, double blind, placebo controlled study, 280 patients were randomly assigned to receive placebo (n = 95) or T-614 at 50 mg (n = 93) or 25 mg (n = 92) daily. Active disease was defined by 4 of the following 5 criteria: >or= 5 tender joints, >or= 3 swollen joints, morning stiffness lasting for >or= 60 minutes, and Westergren erythrocyte sedimentation rate (ESR) >or= 28 mm/h, the assessment of pain at the rest by patient as moderate or severe. Clinical and laboratory parameters were analyzed at baseline, 2, 4, 6, 12, 18 and 24 weeks. The primary efficacy variable at week 24 was the American College of Rheumatology (ACR) response rate using the intent-to-treat population.

RESULTSThe ACR response rate was significantly higher in the T-614 treatment group compared with the placebo group within 8 weeks after the initiation of treatment. After 24 weeks, the 25 mg/d and 50 mg/d dosage groups and the placebo group showed 39.13%, 61.29% and 24.21% in ACR20 and 23.91%, 31.18% and 7.37% in ACR50, respectively. A time-response in ACR response was observed, with clear superiority for the 25 mg/d and 50 mg/d dosage groups compared to placebo (P < 0.0001), and the 50 mg/d dose compared to the 25 mg/d dose (P < 0.05) when using the ACR response analyses after 24 weeks. ESR and c-reactive protein (CRP) were significantly different in the treatment groups after 24 weeks. The incidence of adverse events (AEs) was not significantly higher with T-614 than with placebo, but upper abdominal discomfort, leucopenia, elevated serum alanine aminotransferase (sALT), skin rash and/or pruritus were more common in the 50 mg and 25 mg dosage groups.

CONCLUSIONT-614, a new slow-acting drug, is effective in treatment of rheumatoid arthritis and is well tolerated.

Adult ; Aged ; Antirheumatic Agents ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Benzopyrans ; adverse effects ; therapeutic use ; Double-Blind Method ; Female ; Humans ; Male ; Middle Aged ; Sulfonamides ; adverse effects ; therapeutic use

Objective To examine cerebral pathologies in cerebral amyloid angiopathy in a rat model of Alzheimer's disease. Methods Rat models of Alzheimer's disease was established by stereotactic Aβ1-42 fiber injection in the bilateral hippocampus. The cognitive function of the rats was evaluated with water maze test. HE staining, Congo red staining and double-labeling indirect immunofluorescence were used to examine the dynamic distribution of Aβ fiber deposit in the brain. Results The model rats showed significant differences from the control rats in the escape latency and the times of crossing platform in waster maze test. HE staining revealed a decreased number and degeneration of the granular cells with increased glial cells in the model rats. Congo Red staining showed that the Aβ fiber was deposited gradually in the small vessels in the brain parenchyma to cause thickening, stenosis or occlusion of the small vessels. Immunofluorescence staining detected Aβ fiber migration from the parenchyma to the walls of the small arteries in the rat models. Conclusion Cerebral amyloid angiopathy is a major pathological feature in Alzheimer's disease.

Objective To examine cerebral pathologies in cerebral amyloid angiopathy in a rat model of Alzheimer's disease. Methods Rat models of Alzheimer's disease was established by stereotactic Aβ1-42 fiber injection in the bilateral hippocampus. The cognitive function of the rats was evaluated with water maze test. HE staining, Congo red staining and double-labeling indirect immunofluorescence were used to examine the dynamic distribution of Aβ fiber deposit in the brain. Results The model rats showed significant differences from the control rats in the escape latency and the times of crossing platform in waster maze test. HE staining revealed a decreased number and degeneration of the granular cells with increased glial cells in the model rats. Congo Red staining showed that the Aβ fiber was deposited gradually in the small vessels in the brain parenchyma to cause thickening, stenosis or occlusion of the small vessels. Immunofluorescence staining detected Aβ fiber migration from the parenchyma to the walls of the small arteries in the rat models. Conclusion Cerebral amyloid angiopathy is a major pathological feature in Alzheimer's disease.

BACKGROUNDChronic obstructive pulmonary disease (COPD) has a variable natural history and not all individuals follow the same course. This study aimed to identify the prevalence and characteristics of asymptomatic COPD patients from a population-based survey in China.

METHODSA multistage cluster sampling strategy was used in a population from seven different provinces/cities. All residents (over 40 years old) were interviewed with a standardized questionnaire and spirometry. Post-bronchodilator forced expiratory volume in 1 second (FEV(1))/forced vital capacity (FVC) of less than 70% was defined as the diagnostic criterion of COPD. All COPD patients screened were divided into symptomatic group and asymptomatic group according to the presence or absence of chronic respiratory symptoms. Socio-demographic, personal and exposure variables were collected and analyzed.

RESULTSAmong the 1668 patients who were diagnosed with COPD from the 25 627 sampling subjects, 589 (35.3%) were asymptomatic. The age, sex, body mass index (BMI), rural and urban distributions, smoking habit and education levels were similar in the two groups. A total of 64.7% of the asymptomatic patients had no comorbidities. Cardiovascular diseases and lung cancer were more common among symptomatic COPD patients than asymptomatic group. Asymptomatic COPD group were less likely to present with poor ventilation in the kitchen, a family history of respiratory disease and recurrent childhood cough. Asymptomatic COPD patients had significantly higher FEV(1) (73.1% vs. 61.0%), FVC (91.9% vs. 82.0%), and a higher ratio of FEV(1)/FVC (62.9% vs. 58.7%) (all P < 0.001) than symptomatic group. More asymptomatic patients were underdiagnosed (91.9% vs. 54.3%, P < 0.001) than symptomatic patients.

CONCLUSIONSThis large population-based survey confirmed a high prevalence of asymptomatic COPD patients in China. More use of spirometry screening test may be important to the early detection of COPD.

Adult ; Aged ; Aged, 80 and over ; China ; epidemiology ; Educational Status ; Female ; Humans ; Male ; Middle Aged ; Pulmonary Disease, Chronic Obstructive ; diagnosis ; epidemiology ; Risk Factors ; Smoking ; Spirometry ; Surveys and Questionnaires

BACKGROUNDThe long term prognosis of unprotected left main coronary artery (LMCA) stenting is controversial. This study was conducted to evaluate the immediate and long term outcomes of LMCA stenting in Chinese patients and to determine which factors affect the outcomes.

METHODSFrom May 1997 to March 2003, 224 patients in 23 hospitals underwent elective unprotected LMCA stenting with bare metal stents. Their clinical records were analysed to ascertain immediate and long term outcomes of LMCA stenting as well as factors influencing the prognosis.

RESULTSStents were implanted into LMCA successfully in 223 cases (99.6 %). One death (0.5%) and one case of non-Q wave nonfatal myocardial infarction (MI) occurred in hospital. The mean follow-up time was (15.6 +/- 12.3) months. Cardiac death developed in 10 cases (4.5%), noncardiac death in 2 cases (0.9%), nonfatal MI in 4 cases (1.8%), target lesion revascularization (TLR) of LMCA in 26 cases (11.7%) and TLR of nonLMCA in 37 cases (16.5%). Univariate analysis showed that cardiac death correlated with left ventricular ejection fraction (LVEF < 40%), female gender and LMCA combined with multivessel disease; that major adverse cardiac events (MACE) correlated with LVEF < 40%, bifurcation lesion and incomplete revascularization. Logistic regression analysis revealed that LVEF < 40% and female gender were independent predictors of cardiac death and MACE. Follow-up angiography was performed in 102 cases (45.7%). The restenosis rate was 31.4%.

CONCLUSIONSLong-term outcomes of stenting for selected patients with unprotected LMCA stenosis is acceptable. It should be performed in inoperable or low risk patients with LVEF > or = 40% and isolated LMCA disease or LMCA combined with multivessel diseases in whom complete revascularization can be obtained.

Adult ; Aged ; Aged, 80 and over ; Coronary Angiography ; Coronary Disease ; therapy ; Coronary Restenosis ; etiology ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Stents ; adverse effects ; Treatment Outcome

BACKGROUNDVascular anomalies are common and multidisciplinary involved diseases. The greatest impediment to their treatment in the past was their confusing terminology and clinical heterogeneities. This hospital-based retrospective study assessed some clinical characteristics, diagnosis, therapies and outcomes of patients with vascular anomalies in southeast China.

METHODSA total of 592 vascular anomalies patients (patients with intracranial tissues or viscera involved were excluded), admitted to the First Affiliated Hospital of Sun Yat-sen University from January 2006 to September 2009, were enrolled in the study. Data for clinical characteristics, diagnosis, therapies and outcomes were collected and analyzed.

RESULTSOf the 592 patients, the male:female ratios in the vascular tumor group (n = 187) and the vascular malformation group (n = 405) were 1:1.49 and 1:1.06 respectively, with no significant difference between them. The mean onset age of the vascular tumor group was significantly younger than that of the vascular malformation group (p < 0.001). The head and neck were the most commonly (31.4%) involved areas in vascular anomalies. A total of 23.8% of the patients with vascular anomalies had definite symptoms caused by the vascular lesions. In the vascular tumor group, 94.1% of them were infantile hemangiomas. Venous malformation was the most common (41.0%) subtype of vascular malformations. Surgical therapy was undertaken in 94.2% of the patients with vascular anomalies. Of the 519 patients available for the 16 - 58 month follow-up, 322 patients (62.0%) were cured, 108 patients (20.8%) were markedly improved, 57 patients (11.0%) were partially improved, and 32 patients (6.2%) were uncured.

CONCLUSIONSVascular anomalies are clinically heterogeneous. While the outcome is generally favorable, further effort should be made to determine the appropriate terminology and management.

Adolescent ; Blood Vessels ; abnormalities ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Retrospective Studies ; Vascular Neoplasms ; epidemiology

BACKGROUNDAlthough thrombolytic therapy with rescue percutaneous coronary intervention (PCI) is a common treatment strategy for ST-segment elevation acute myocardial infarction (STEMI), scant data are available on its efficacy relative to primary PCI, and comparison was therefore the aim of this study.

METHODSThis multicenter, open-label, randomized, parallel trial was conducted in 12 hospitals on patients (age < or = 70 years) with STEMI who presented within 12 hours of symptom onset (mean interval > 3 hours). Patients were randomized to three groups: primary PCI group (n = 101); recombinant staphylokinase (r-Sak) group (n = 104); and recombinant tissue-type plasminogen activator (rt-PA) group (n = 106). For all patients allocated to the thrombolytic therapy arm, coronary angiography was performed at 90 minutes after drug therapy to confirm infarct-related artery (IRA) patency; rescue PCI was performed in cases with TIMI flow grade < or = 2. Bare-metal stent implantation was planned for all patients.

RESULTSAfter randomization it required an average of 113.4 minutes to start thrombolytic therapy (door-to-needle time) and 141.2 minutes to perform first balloon inflation in the IRA (door to balloon time). Rates of IRA patency (TIMI flow grade 2 or 3) and TIMI flow grade 3 were significantly lower in the thrombolysis group at 90 minutes after drug therapy than in the primary PCI group at the end of the procedure (70.5% vs. 98.0%, P < 0.0001, and 53.0% vs. 85.9%, P < 0.0001, respectively). Rescue PCI with stenting was performed in 117 patients (55.7%) in the thrombolytic therapy arm. Rates of patency and TIMI flow grade 3 were still significantly lower in the rescue PCI than in the primary PCI group (88.9% vs. 97.9%, P = 0.0222, and 68.4% vs. 85.0%, P = 0.0190, respectively). At 30 days post-therapy, mortality rate was significantly higher in the thrombolysis combined with rescue PCI group than in primary PCI group (7.1% vs. 0, P = 0.0034). Rates of death/MI and bleeding complications were significantly higher in the thrombolysis with rescue PCI group than in the primary PCI group (10.0% vs. 1.0%, P = 0.0380, and 28.10% vs. 8.91%, P = 0.0001, respectively).

CONCLUSIONSThrombolytic therapy with rescue PCI was associated with significantly lower rates of coronary patency and TIMI flow grade 3, but with significantly higher rates of mortality, death/MI and hemorrhagic complications at 30 days, as compared with primary PCI in this group of Chinese STEMI patients with late presentation and delayed treatments.

Aged ; Angioplasty, Balloon, Coronary ; Coronary Angiography ; Female ; Fibrinolytic Agents ; therapeutic use ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; drug therapy ; therapy ; Thrombolytic Therapy