This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.

OBJECTIVETo investigate the distribution and role of alpha, beta and gamma subunits of epithelial sodium channel (ENaC) in the cochlea and endolymphatic sac of guinea pig.

METHODSThe expression of alpha-, beta- and gamma-ENaC subunits proteins was studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the alpha, beta and gamma subunits of rat ENaC. Alpha-ENaC mRNA was detected by in situ hybridization with digoxin labeled cDNA probe.

RESULTSAll three subunits of ENaC, alpha-, beta- and gamma-, were widely distributed in the labyrinth. In the cochlea, strong labeling of alpha-ENaC protein was found in the spiral limbus, and to a less extent, in the spiral ligament, organ of Corti and Reissner's membrane. The immunoreactivity of beta-ENaC was observed in the spiral ligament, spiral limbus, spiral ganglion, organ of Corti and Reissner's membrane with a less intensity than that of alpha-ENaC. Gamma-ENaC was presented primarily in the superior part of the spiral ligament, spiral limbus, spiral ganglion, and weakly in the organ of Corti and Reissner's membrane. In the endolymphatic sac, intensive immunoreactivities of all three subunits were seen in the epithelial cells and the subepithelial cells at similar intensity. Alpha-ENaC mRNA was localized in the spiral limbus, the inferior part of spiral ligament, stria vascularis, and epithelial cells and subepithelial cells of endolymphatic sac.

CONCLUSIONDifferent subunits of the ENaC expressed in various cell regions of the cochlea and endolymphatic sac in distinct patterns may form the functional sodium channel to regulate the endolymph, thus serve to maintain homeostasis in inner ear.

Animals ; Cochlea ; metabolism ; Endolymphatic Sac ; metabolism ; Epithelial Sodium Channels ; metabolism ; Guinea Pigs

OBJECTIVETo investigate the biomechanical effect of different volume, distribution and leakage to adjacent disc of bone cement on the adjacent vertebral body by three-dimensional osteoporosis finite element model of lumbar.

METHODSL(4)-L(5) motion segment data of the cadaver of an old man who had no abnormal findings on roentgenograms were obtained from computed tomography (CT) scans. Three-dimensional model of L(4)-L(5) was established with Mimics software, and finite element model of L(4)-L(5) functional spinal unit (FSU) was established by Ansys 7.0 software. The effect of different loading conditions and distribution of bone cement after vertebroplasty on the adjacent vertebral body was investigated.

RESULTSThis study presented a validated finite element model of L(4)-L(5) FSU with a simulated vertebroplasty augmentation to predict stresses and strains of adjacent untreated vertebral bodies. The findings from this FSU study suggested the endplate and disc stress of the adjacent vertebral body was not influenced by filling volume of bone cement but unipedicle injection and leakage to the disc of bone cement could concentrate the stress of adjacent endplate.

CONCLUSIONSAsymmetric distributions and leakage of cement into intervertebral disc can improve the stress of endplate in adjacent vertebral body. These results suggest that optimal biomechanical configuration should have symmetric placement and avoid leakage of cement in operation.

Bone Cements ; adverse effects ; pharmacology ; Finite Element Analysis ; Humans ; Imaging, Three-Dimensional ; Lumbar Vertebrae ; surgery ; Male ; Polymethyl Methacrylate ; adverse effects ; pharmacology ; Stress, Mechanical

Humans ; Glucocorticoids/therapeutic use* ; Cochlear Implantation

Objective To determine the effect of Tenuigenin (TEN) on the Mashl expression in neural stem cells (NSCs) from mouse hippocampus and directional differentiation of its NSCs in vitro. Methods NSCs were generated from the mouse hippocampus within 24 hours after birth and cultured with EGF and bFGF (20 ng/mL, each) in a 50-mm uncoated culture flask. The third-passage NSCs were cultured with TEN medium and divided them into vehicle, 5 mg/L, 20 mg/L and 100 mg/L groups according to different TEN concentrations. Immunocytochemical techniques were employed to detect the NSE-positive neurons and ChAT-positive neurons induced by TEN, and the expression of Mash1 in NSCs affected by TEN was measured by RT-PCR techniques. Results The immunocytochemical and RT-PCR results revealed that ChAT-positive neurons and NSE-positive neurons were detected with statistical significance and 20 mg/L group showed a stronger effect than other concentration groups (P< 0.05). The expression of Mash1 in the 20 mg/L group increased as compared with that in the vehicle group (P<0.05). Conclusion TEN can induce NSCs in vitro differentiating into neurons and cholinergic neurons, and increase the expression of Mashl in NSCs.

Objective To study the effect oftenuigenin (TEN) on the processes of neural stem cells (NSCs) injured by β-amyloid (Aβ) protein. Methods Mouse NSCs were generated from the hippocampi of Kunming mice within 24 hour from birth and cultured with epidermal growth factor (EGF)and basic fibroblast growth factor (bFGF) (20 ng/mL each) in 50-mm uncoated culture flasks.The third passage NSCs were cultured in Aβ medium (12.5 μmol/L) and TEN medium (5 mg/L,20 mg/L,100mg/L) respectively and observed under a scanning electron microscope (SEM) after 3 days.Optical microscopy was used to detect the length and amount of the processes of NSCs. The statistical significance between group comparisons was determined by t test.P value ＜0.05 was considered to be statistically significant. Results The length and amount of NSC processes in Aβ1-42 group were both significantly shorter and smaller than in the control group (P＜0.05). The length and amount of NSC processes in 20 mg/L and 100 mg/L groups were both significantly longer and larger than in the Aβ-42group (P＜0.05). Conclusion TEN can significantly increase the length and amount of NSC processes injured by Aβ.

The hydration reaction by microbial method is the crisis of the procedure of acrylamide production from acrylonitrile. This research studied the enzyme catalytic kinetics and de-active kinetics of nitrile hydratase in the type of free cell. Firstly, the effects of the concentration of cells, the temperature, pH value, the concentration of acrylonitrile and the concentration of acrylamide on the activity of nitrile hydratase was investigated. The result is that the temperature and the concentration of acrylamide are the most important among these factors. The activity of the nitrile hydratase was 5659 u/mL (broth) at 28 degrees C; the counterpart was only 663 u/mL (broth) at 5 degrees C. And the activity of NHase in solution of 45% acrylamide was just about half of that in solution of 5% acrylamide. After study on the relation of temperature and the reaction speed, It was found that the activation energy of the hydration of NHase was 65.57 kJ.mol-1. This paper studied the effects of concentration of cells, temperature, pH value, concentrations of acrylonitrile and acrylamide on the deactivation of Nhase, as well as the related enzyme de-active kinetics. The result also showed that the temperature and the concentration of acrylamide are the most important among these factors. In solution of 35% acrylamide, the residual activity was about 0% of the original value after 55 h; but in solution of 10% acrylamide, after the same period of time, the residual activity was 50% of the original one. It was also found that the concentration of acrylonitrile had little effect on the stability of NHase. The coefficient of deactivation at 28 degrees C was 21.77 times of the one at 5 degrees C. Correlating the temperature and the coefficient of deactivation, the activation energy of the de-active reaction was found to be 92.28 kJ.mol-1.
Acrylamide ; metabolism ; Acrylonitrile ; metabolism ; Catalysis ; Hydro-Lyases ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Rhodococcus ; enzymology ; Temperature

The cultural conditions for the growth of Norcardia cell were studied in this paper. Controlling pH value, adding nutrient and optimizing the quantity of inducer during cultivation, the activity of nitrile hydratase reached 6567 u/mL (culture medium), which was the highest value appeared in native journals. In the farther hydratase experiments, no by-product, crylic acid, was detected. It showed that the activity of amidase was not promoted obviously while the activity of nitrile hydratase was increased greatly. The results set a strong foundation for the industrial application and the research on new technology.
Acrylamides ; metabolism ; Amidohydrolases ; metabolism ; Biotechnology ; methods ; Cell Culture Techniques ; Fermentation ; physiology ; Glucose ; metabolism ; Hydro-Lyases ; metabolism ; Hydrogen-Ion Concentration ; Nocardiaceae ; enzymology ; metabolism

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.
D-Amino-Acid Oxidase ; genetics ; Fermentation ; Methanol ; metabolism ; Phenylalanine ; metabolism ; Pichia ; genetics ; Polymerase Chain Reaction

BACKGROUNDThis work was carried out to evaluate the clinical efficacy and the complications of S-ROM modular hip arthroplasty combined with transverse subtrochanteric shortening for Crowe type IV congenital dislocation of the hip (CDH).

METHODSA total of 28 consecutive patients with Crowe type IV CDH received treatment using this surgical technique from June 2003 to June 2010. The follow-up was conducted at 3 days, 1, 6, and 12 months after the operation and later annually at the outpatient of our hospital. Sequential pelvic plain film and normotopia film of the affected hip joint were taken. The limp and the Trendelenburg sign were also assessed, the ischiadic nerve injury was also evaluated by electromyogram, and Harris hip scores were recorded.

RESULTSAfter operation, both the alignment and the position of the transverse osteotomies were good. None of the patients had presented complications of joint infection, prosthesis loosening, joint dislocation, or nerve injury.

CONCLUSIONSS-ROM modular hip arthroplasty combined with transverse subtrochanteric shortening was a satisfactory and safe technique for the Crowe type IV congenital hip dislocation within a mean follow up of 53 months. Transverse subtrochanteric shortening could effectively prevent the distraction injury of sciatic nerve.

Adult ; Arthroplasty ; methods ; Female ; Hip Dislocation, Congenital ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Osteotomy ; methods ; Radiography