In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids.
Cell Line ; DNA Transposable Elements ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Plasmids ; Polymerase Chain Reaction ; Telomerase ; genetics ; Transfection

Objective To explore the clinical diagnosis and treatment of cyclic vomiting syndrome in children.Methods Thirty children proved with cyclic vomiting syndrome admitted from January,1998 to January,2003 were analyzed retrospectively.Results Cyclic vomiting syndrome was most likely to occur in 3-12 years old.The male to female ratio was 3∶2.The clinical manifestations were recurrent vomiting.Twenty-one cases had inducements,while 9 cases had not inducements.It was safe and efficient that curing cyclic vomiting syndrome with cyprohetadine and amitriptyline.Conclusions If these children with cyclic vomiting syndrome are inefficient to treatment,excluding metabolizable diseases,gastrointestinal,neurological diseases,they may be diagnosed cyclic vomiting syndrome,and cured with cyprohetadine and amitriptyline.

The author introduces the basic concepts of microsatellite and population genetics and its characteristics, expounds the application of these theories for population genetic structure and genetic diversity, gene flow and evolutionary significant unit ESU division research. This paper discuss its applicationin study of genetic causes, origin of cultivation, different regional origins of geoherbs, aiming at providing a new theory and method for geoherbs.
Drugs, Chinese Herbal ; Evolution, Molecular ; Genetic Markers ; genetics ; Genetic Techniques ; Geography ; Microsatellite Repeats ; genetics

Objective To investigate the effects of rehabilitation training on hippocampal amyloid-beta peptide (Aβ) and insulin-degrading enzyme (IDE) levels in vascular dementia (VD).Methods Thirty female Sprague-Dawley rats were randomly assigned to a rehabilitation group (n =10),a model group (n =10) or a sham-operation group (n =10).An experimental VD model was established in the rats of the first 2 groups by bilateral common carotid artery permanent ligation.The rats in the rehabilitation group then received 1 h of rehabilitation training daily.Learning and memory were assessed at 4 weeks aftet the operation.Immunohistochemical staining was used to detect Aβ and IDE expression in the hippocampus dentate gyrus (DG) area.Results The rats in the rehabilitation group showed significantly better learning ability compared with the model group.The expression of Aβ in the rehabilitation group was significantly less than in the model group.The expression of IDE in the rehabilitation group was significantly greater Conclusion Rehabilitation can accelerate the recovery of learning and memory in VD,at least in rats The mechanism is possibly related to decreased accumulation of Aβ in the hippocampus due to up-regulation of the expression of IDE.

ObjectiveTo study the effect of exercise training on β-amyloid polypeptide (Aβ) and β-secretase(BACE) in the hippocampus of the rats with vascular dementia (VD).Methods 30 Sprague-Dawley (SD) rats were carried out to an exercise group (n =10 ),a model group (n =10 ),and a sham-operation group ( n =10 ).VD rat models were made by the ligation of bilateral common carotid arteries.Morris water maze test were carried out 4 weeks after the operation to assess the ability in learning and memory of the rats and Aβ and β-secretase (BACE)expression was detected in the hippocampus of the rats using immunohistochemical techniques.ResultsIn the Morris water maze test,the model group showed reduction in the learning and memorizing ability,with obvious longer escape latencies ( ( 101.34 ± 19.67 ) s,(95.42 ± 23.89 ) s,( 89.39 ± 22.67 ) s,( 90.12 ± 19.77 ) s,respective-ly) than that of sham-operation group ( ( 62.13 ± 11.38 ) s,( 24.84 ± 13.69 ) s,( 16.98 ± 12.51 )s,( 11.41 ± 8.93 ) s,correspond-dingly) (P ＜ 0.05 ),and the exercise group was improved in the learning and memorizing ability ( corresponding to ( 80.15 ± 21.56 ) s,( 51.24 ± 20.91 ) s,( 43.78 ± 22.36) s,( 45.67 ± 20.87 ) s ),compared with the model group(P＜0.05).The grey values of Aβ in the hippocampus of the rats for the exercise group was ( 130.12 ± 19.01 ),( 116.77 ± 23.67 ) for the model group and ( 148.44 ± 17.67 ) for the sham-operation group(P＜ 0.05).The grey values of BACE in the hippocampus of the ratsfor the exercise group were( 131.21± 25.25 ),( 120.53± 10.21 ) for the model group(P＜ 0.05 ) and ( 162.38 ± 28.11 ) for the sham-operation group (P ＜ 0.05).ConclusionExercise training can lower the expression of BACE and Aβ in the hippocampus of rats with VD,therefore improving the learning and memory ability of rats with VD.

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.
Cloning, Molecular ; DNA, Complementary ; genetics ; Diterpenes, Abietane ; biosynthesis ; Enzymes ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Salvia miltiorrhiza ; enzymology ; genetics

To evaluate the efficacy of Tuina and Chinese patent drug Shuxuetong injection in preventing patients undergoing total knee arthroplasty from deep venous thrombosis and in functional rehabilitation.

OBJECTIVETo study the change in expressions of nerve growth related protein (GAP-43) and neurofilament (NF) after experimental injury of spinal cord, and the regulatory effect of Chinese medicine Suifukang (SFK) on them.

METHODSForty-eight from the 54 selected adult SD rats were established into spinal cord injury model by making hemitransection at the T12 level, and randomly divided into two groups, the SFK group feed with SKF contained liquor and the model group feeding with equal volume of saline. The another 6 rats were untreated and taken as the normal group. All rats were sacrificed in batches at different time points of day 3, 7, 15 and 30 after modeling. The spinal cord was obtained for determining the optic density (OD) of positive expression of GAP-43 and NF with immunohistochemical stain by microscopic and semi-quantitative image analysis.

RESULTS(1) OD of NF in the model group was obviously decreased on day 7, showing significant difference to that in the normal group and that in the SFK group (P < 0.05), and it maintained at low level after then, while the OD of NF in the SFK group was obviously higher than that in the model group (P < 0.05), it arrived the peak on day 15 and then dropped near the level in the normal group on day 30; (2) OD and of GAP-43 in the model group obviously decreased on day 3 (P < 0.05), showing significant difference to that in the normal group (P < 0.05), then it returned to approach to the level in the normal group on day 7. It was higher in the SFK group than in the model group on day 3, and maintained the high level to day 7, at that time, it was still higher than that in the model group and also higher than that in the normal group (P < 0.05).

CONCLUSIONSFK can promote the repair and regeneration of injured nerve fibers in spinal cord by up-regulating the expression of GAP-43 and NF.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; GAP-43 Protein ; biosynthesis ; Immunohistochemistry ; Male ; Nerve Regeneration ; drug effects ; Neurofilament Proteins ; biosynthesis ; Neuroprotective Agents ; therapeutic use ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; drug therapy ; metabolism ; physiopathology

Four strains of aerobic denitrifying bacteria were isolated from biomembrane of the laboratory biofilter by selective medium. The denitrifying rates of these four strains were found more than 80% under aerobic condition for 40 hours. The denitrifying rate of A1 was highest, which was 99.05%. When measuring the course of nitrogen element changing, it is found that they accumulated nitrite expect the strain A1. The strain A1 was gram positive and spherical. It is identified as Pseudomonas putida based on its biochemical and morphological characters and phylogenetic analysis of 16S rDNA sequences(genbank accession NO.DQ836052.1). For the strain A1, the optimum beginning pH was 7.0 around, and the optimum tempera- ture was 30 ℃ around, The change of DO did not influence the effect of denitrification when it was more than 2.0 mg/L.

A stable and efficient L-Malic acid accumulation mutant strain Rhizopus oryzae ME-M15 was discovered occasionally in the mutation breading for fumaric acid producers. Rhizopus oryzae ME-M15 gave a L-Malic acid output of 16.3 g/L on average after fermentation for 96 hours, more than 3 times than that of the parent strain ME-F10. In addition, other metabolites such as ethanol and fumaric acid were re-markably decreased in accordance with the depressed activity of the cytosolic isoenzyme of fumarase and alcohol dehydrogenase in strain ME-M15, while the activity of the pyruvate carboxylase had no significant difference.