The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.

Objective To identify seven species of Gynostemma BI.,including G.pentaphyllum,G. pentagynum,G.cardiospermum,G.longipes,G.yixingense,G.laxiflorum,and G.guangxiense,by in- ter-simple sequence repeat(ISSR)markers.Methods General DNA was isolated from leaves of the seven species in Gynostemma B1.by CTAB,57 primers constituted by ISSR were tested for PCR and sepharose electrophoresis.Results Fourteen primers amplified polymorphic bands,the amplification patterns of primers UBC-873 and UBC-895 were higher in terms of polymorphic and amplified band ratio.They are used to distinguish all the examined seven species.Conclusion ISSR-PCR Method provides a quick,reli- able molecular marker technique for the identification of different species of Gynostemma B1.

A simple and cost-effective indirect competitive enzyme-linked immune sorbent assay (ic-ELISA) was developed to rapidly screen the content of aflatoxin B1 (AFB1) in lotus seeds, and the results were confirmed by ultra-fast liquid chromatography-tandem mass spectrometry( UFLC-MS/MS). Matrix-matched calibration expressed a good linearity ranging from 0. 171 to 7. 25 µg · L(-1) for AFB, with R2 > 0.978. The medium inhibitory concentration( IC50 ) for AFB1 was 1.29 µg · L(-1), the recovery for AFB1 was 74.73% to 126.9% with RSD < 5%, and the limit of detection (IC10) was 0.128 µg · L(-1). The developed ic-ELSIA method was applied to rapid analysis of AFB, in 20 lotus seeds samples and the results indicated that the contents of AFB, in samples 1-15 were in the range of 1. 19- 115. 3 µg · kg(-1) and in 40% of the samples exceeded the legal limit(5 µg · kg(-1)), while the contamination rate of AFB, in samples 16-20 was 40%. Pearson correlation coefficient(r) reached 0.997 for AFB1 content in the samples detected by ic-ELSIA and UFLC-MS/MS methods. The results proved that the developed ic-ELISA method is simple, sensitive and reliable, and can be used for rapid and high-throughput screening of AFB1 in lotus seeds
Aflatoxin B1 ; analysis ; Drug Contamination ; Enzyme-Linked Immunosorbent Assay ; methods ; Loteae ; chemistry ; Seeds ; chemistry

OBJECTIVEAccidents are an important cause of childhood injury. It is hypothesized that safety education programs can reduce accidents in primary school-aged children. This study aimed to determine whether child and parent safety education programs can decrease the incidence of accidental injury in children when compared with controls.

METHODSThe study population (aged 7-13 years) were recruited from four local primary schools, and randomly assigned into an Intervention or a Control group. The Intervention group received child and parent safety education and was taught injury prevention strategies. The Control group received no injury prevention education or intervention. The incidence of accidental injury was compared between the two groups.

RESULTSIn the first year after intervention the incidence of accidental injury was 262 cases in the Intervention group (8.26%) and 234 cases (8.67%) in the Control group (P > 0.05). In the second year after intervention, however, the incidence of accidental injury was significantly less in the Intervention group (211 cases, 6.54%) compared with the Control group (229 cases, 8.63%) (P < 0.01).

CONCLUSIONSInjury prevention strategies and child and parent safety education can reduce risks of accidental injury in children.

Accident Prevention ; methods ; Adolescent ; Child ; Female ; Humans ; Male ; Parents ; Safety ; Wounds and Injuries ; prevention & control

Objective To investigate the neuroimaging features in mitochondrial encephalomyopatbies with lactic acidosis and stroke-like episodes(MELAS).Methods Twenty-two clinically diagnosed patients who came from department of neurology,Huashan hospital in October 2003 to July 2006 were analyzed for CT,MRI,MRI contrast,MRA and MRS.Results In all 22 patients,the neuroimaging results of 21 were positive.There were 9 patients lying in hemisphere,12 in both cerebral hemispheres,including occipital,parietal,temporal and frontal lobe.The abnormal areas showed low signal intensity on T_1-weighted MRI,high signal intensity on T_2-weighted MRI and fluid attenuated inversion recovery(FLAIR)images.The lesions of 12/16 patients on MR contrasted images were enhanced.The lesions of one patient showed malacoma-like changes,one showed Fahr syndrome' s change and another showed high signal intensity on MR contrasted images.Conclusion Although the neuroimaging features of MELAS are complicated,the specific ones could help to make the diagnosis.

AIM To observe the curative effects of Modified Buzhong Yiqi Decoction (Codonopsis Radix,Astragali Radix,Atractylodis macrocephalae Rhizoma,ect.) on perioperative period of intertrochanteric fractures in elderly patients with Qi deficiency and blood stasis type.METHODS One hundred and twenty cases of elderly intertrochanteric fractures were randomly divided into treatment group and control group,60 cases in each group.All patients underwent closed fracture reduction and proximal femoral nail anti-rotation (PFNA) fixation.During the perioperative period,the control group only received conventional treatment,while the treatment group received conventional treatment and Modified Buzhong Yiqi Decoction once a day.The levels of interleukin-6 (IL-6),interleukin-10 (IL-10) and tumor necrosis factor (TNF-α) of both groups in preoperative and in the 1st day,the 7th day,the 14th day of postoperative were observed and compared.The complications and the curative effects according to the Harris score also were observed and compared between the two groups.RESULTS In preoperative,there was no significant difference in levels of IL-6,IL-10 and TNF-α between the two groups (P ＞ 0.05).The levels of IL-6 and TNF-α in the 1 st day of postoperative in the control group were significantly higher than those in the treatment group (P ＜0.05,P ＜0.01).And the levels of IL-10 were similar between the two groups (P ＞0.05).In addition,in the 7th day and the 14th day of postoperative,the levels of IL-6,IL-10 and TNF-α in both groups were all decreased,moreover,the descent degree in treatment group was more significant than those in the control group (P ＜ 0.05,P ＜ 0.01).The treatment group had the lower incidence of complications and the higher curative effects than those in the control group,both differences were statistically significant (P ＜0.05).CONCLUSION Modified Buzhong Yiqi Decoction applying to the perioperative period of femoral intertrochanteric fractures in elderly patients with Qi deficiency and blood stasis,can effectively reduce the perioperative inflammatory response,reduce the incidence of postoperative complications,and improve the curative effects.So it has a positive effect on the rehabilitation for the traumatic patients and is worthy of clinical promotion.

To study the effects of sodium valproate (VPA) on human myelodysplastic syndrome cell line MUTZ-1. The cell proliferation was determined by MTT assay, apoptotic morphological features were observed by light microscopy and transmission electronmicroscopy, cell apoptosis and cell cycle shift were analyzed by flow cytometry (FCM). The results showed that VPA could inhibit the growth of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in MUTZ-1 cells treated with 4 mmol/L VPA for 72 hours. Pyknosis of cells and nuclei, disintegration of nuclear chromatin and apoptotic body could be observed by light microscopy. Aggregation and margination of nuclear chromatin, concentration of plasm, increment of density and chromatin mass of irregular size could be observed by transmission electronmicroscope. The flow cytometric analysis indicated that the VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner, ratio of cells at G(0)/G(1) phase increased and ratio of cells at S phase decreased in dose-dependent manner, the cells were arrested at G(0)/G(1) phase. It is concluded that the VPA can induce apotosis and inhibite proliferation of MUTZ-1 cells via arresting cells at G(0)/G(1) phase.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Humans ; Myelodysplastic Syndromes ; pathology ; Valproic Acid ; pharmacology

To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Animals ; Ciliary Neurotrophic Factor ; genetics ; Humans ; Mice ; Mutant Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; genetics

AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Ciliary Neurotrophic Factor ; biosynthesis ; genetics ; Genetic Vectors ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Mutation ; Peptide Hydrolases ; chemistry ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUND: Umbilical cord mesenchymal stem cells (UC-MSCs) are a kind of pluripotent stem cells capable of relieving inflammation and edema in injured areas, promoting microvascular regeneration and improving wound healing. OBJECTIVE: To explore the therapeutic effects of UC-MSCs transplantation on acute traumatic brain injury (TBI) in rats and the effect on microvessel density of the rat brain tissue. METHODS: Thirty Sprague Dawley rats were randomly divided into three groups, control group, TBI model group and UC-MSCs treatment group (n=10 per group). TBI models were made in the rats by free fall method. US-MSCs (1.5×106) were infused through the cerebral ventricle in the rats. Neurological severity scores were assessed at 24 hours after cell transplantation. The microvessel density in the injured brain tissue was detected using immunohistochemistry method at 14 days after cell transplantation. RESULTS AND CONCLUSION: Compared with the TBI model group, the microvessel density value was significantly increased after UC-MSCs transplantation (P < 0.05), and the neurological severity score also became better in the UC-MSCs treatment group (P < 0.05). In summary, UC-MSCs transplantation can effectively improve became vascular remodeling and have a good neuroprotection in acute TBI rats.