The purpose of this study is to investigate the enantioselectivity of norgestrel (NG) transdermal permeation and the potential influence of linalool and lipids on the enantioselectivity. In vitro skin permeation studies of NG across the excised rat skins were performed with Valia-Chien diffusion cells, and the permeation samples were analyzed by enantioselective HPLC. The possible enantioselective permeation of NG across intact rat back skin and lipids extracted rat back skin and the influence of linalool were evaluated. The skin permeation rate of dl-NG was two times higher than that of l-NG when donor solutions (EtOH/H2O 2 : 8, v/v) containing l-NG or dl-NG. It may be mainly attributed to the solubility discrepancy between enantiomer and racemate. The enantioselective permeation of dl-NG across intact rat skin was observed when the donor solutions containing dl-linalool. The permeation flux of l-NG was 22% higher than that of d-NG. But interestingly, the enantioselective permeation of dl-NG disappeared under the same experimental condition except that the lipid extracted rat skin was used. Attenuated total reflection-fourier transform infrared spectroscopy analysis of stratum corneum showed that the wave number for asymmetric CH2 stretching vibrations of lipids treated with dl-linalool was greater than that of the control. The results indicated that the enantioselective permeation of NG may be contributed by the interaction between dl-linalool and lipids. More than half of lipids were composed of ceramides. The stereospecific interaction maybe existed among chiral enhancer (linalool), lipids (ceramides) and/or chiral drugs (NG).
Administration, Cutaneous ; Animals ; Lipids ; pharmacology ; Monoterpenes ; pharmacology ; Norgestrel ; pharmacokinetics ; Rats ; Skin Absorption ; drug effects ; Spectroscopy, Fourier Transform Infrared ; Stereoisomerism

Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10％ fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5％,5.0％,l0.0％,15.0％ and 20.0％,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10％ FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0％ aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P ＜ 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)％ in the 10.0％ aqueous humors group and (23.06±1.13)％ in the control group,showing a significant difference (t =-5.729,P=0.005).The scratch test showed that the healing area of the cell signal layer was (0.116±0.019) mm2 in the 10.0％ aqueous humors group and (0.358 ±0.049) mm2 in the control group,showing a significant difference (t =13.842,P =0.000).The density of cells in the 10.0％ aqueous humor group was (1439± 1 10)/field,which was more than (1162±45)/field in the control group (t =-11.020,P=0.000).Conclusions Aqueous humor at the concentration of 10.0％ promote the growth and proliferation of bovine CECs.The result suggests that 10.0％ aqueous humor can be used as a promoting agent during the culture of CECs.

Aim Toresearchthemolecularmecha-nisms of DADS-induced apoptosis in human leukemia K562cells.Methods Cellviabilitywasmeasuredby MTT. Levels of DADS-induced ROS were measured by 2ˊ, 7ˊ-dichlorofluorescein diacetate ( DCFH-DA) fluo-rescence. DADS-induced mRNA levels of components of the NADPH oxidase were detected by Real-time PCR. The combination of protein Rac2 and p67phox was measured by immunoprecipitation assays. Flow cy-tometry methods were used to determine the percentage of apoptosis cells. DADS-induced Rac2 levels were measuredbyWesternblot.Results TheDADS-trea-ted K562 cells showed a dose-and time-dependent de-crease in cell viability and proliferation. There was sig-nificant up-regulation of the mRNA level of components of the NADPH oxidase complex in K562 cells after treatment with 6 mg·L-1 DADS for 6 h. Western blot results revealed that, compared with the control group, there was a significant up-regulation of Rac2 protein in K562 cells treated with 5. 0 and 10. 0 mg·L-1 DADS for 24h. And Rac2 combined with p67phox in DADS-induced apoptosis in K562 cells. PMA markedly in-creased the percentage of apoptotic cells, and DPI re-duced the percentage of apoptotic cells in DADS-in-duced K562 cells. Levels of DADS-induced ROS, also showed enhancement when exposed in PMA, but there was no DADS-induced ROS production evident when exposed in DPI in DADS induced K562 cells. Conclu-sions TheseresultsindicatethatNADPHoxidaseis the main source of DADS-induced ROS production. Diallyl disulfide induces apoptosis in human leukemia K562 cells through activation of NADPH oxidase.

Background Induced pluripotent stem cells (iPSCs)can differentiate into various types of somatic cells without causing ethical controversy and immune rejection in clinical activity,which is similar to differentiation ability of embryonic stem cells.So,iPSCs may be used as seed cells for tissue engineering corneal endothelial reconstruction.Objective The present study was to survey the morphologic change of iPSCs after coculture with corneal endothelium cells(CECs) under the atomic force microscopy(AFM).Methods Rabbit CECs and human MMC-iPSCs were isolated and cultured respectively.The iPSCs were identified with the marker by immunochemistry.iPSCs passaged for 7 days were then cultured with 60％ confluent CECs to establish the co-culture model.The surface morphology and cellular membrane ultrastructure of differentiated iPSCs after induced by CECs were examined by AFM combination with inverted microscope,and compared with CECs and undifferentiated iPSCs.Results Thelengthand width were(66.93±10.48)μm and (44.85 ± 8.14) μm in CECs,(12.51±1.40)μm and (10.93 ±1.69) μm in uninduced iPSCs,and(36.12±10.29) μm and(31.53±9.65)μm in CECs-induced iPSCs.Both the length and width values of CECs-induced iPSCs were statistically bigger than those uninduced iPSCs,with significant differences between them (P＜0.05),but no significant difference was seen in the width valne of CECs-induced iPSCs in comparison with CECs(P＞0.05).The convex structure of CECs cytomembrane surface showed the digitation in shape with the size and height(2.11 ± 1.03) μm and (115.68±92.08) nm respectively,and the concave structure of cytomembrane surface of CECs was fenestrae-like depression and the size was (1.49 ± 0.65) μm.The numerical valuc of mean square root roughness (Rq)and average roughness (Ra)of cytomembrane surface of CECs were(39.20±7.82)nm and (30.37±5.32)nm respectively.The convex surface of cytomembrane of iPSCs was granular-like in shape with size and height(0.39±0.22)μm and(13.11±9.18)nm respectively.The concave surface of cytomembrane of iPSCs was worm-eaten-like concave with the size(0.34±0.18)μm.The numerical value of Rq and Ra of geometrical parameters of cytomembrane surface of iPSCs were (26.60 ± 4.93)nm and (9.97 ± 3.78) nm respectively.The convex surface of cytomembrane of induced iPSCs was digital-like in shape with the size and height (1.91±0.76) μm and(106.55±77.27) nm respectively.The concave surface of cytomembrane of induced iPSCs was fenestrae-like depression and the size of concave was(1.6l±1.25) μm.The numerical value of Rq and Ra on surface of cytomembrane of induced iPSCs was (57.33± 12.80) nm and (43.63± 11.17) nm respectively.The numerical values of the size and height of convex,the size of concave,Rq and Ra on surface of cytomembrane in induced iPSCs were statistically bigger than in iPSCs(P＜0.05)and were not significant differences in comparison with CECs (P＞0.05).Conclusions Morphology of iPSCs translate toward the CECs after induce for 7 days under the AFM.This outcome lays the foundation for further study on iPSCs.

Background The multipotent differentiation features of induced pluripotent stem cells (iPSCs) offer a new option for cell replacement therapy of many clinical diseases.In ophthalmology,iPSCs are a good model in studying the pathogenic mechanism of degenerative ocular diseases.A better identification method for iPSCs is critical for analyzing the in vivo biological characteristics of iPSCs.Objective This study was to investigate the feasibility and stability of labeling iPSCs with quantum dots.Methods Human umbilical mesenchymal stromal cells-iPSC lines were cultured and amplified on matrigel,and the characteristics of iPSCs were evaluated by immunofluorescence.Different concentrations (5.0,7.5 and 10.0 nmol/L) of quantum dots with a CdSe/ZnS nuclear shell structure were used to label iPSCs after passaging and proliferation.The labeling outcome was observed with a three-dimensional deconvolution real-time live cells imaging system.The labeled iPSCs were subsequently cultivated,and then changes in fluorescence intensity were examined 7 days after the first and the second passaging of iPSCs.Results iPSCs were observed to grow in a clonal manner under the inverted microscope.The iPSC markers,OCT4 and Nanog,were detected by immunofluorescence.With increasing concentrations of quantum dots,the fluorescence intensities representing the levels of OCT4 and Nanog in iPSCs were gradually elevated,with optimal levels of fluorescence observed at a concentration of 10 nmol/L of quantum dots.The fluorescent labeling of OCT4 and Nanog in iPSCs remained and weakened gradually till day 7 even after the second passage.Conclusions Quantum dots labeling could be used to track iPSCs in a dose-independent manner.The fluorescent signal from the quantum dots labeling the iPSCs lasts 2 weeks at least.

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; Glycosylation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo evaluate the clinical and radiographic results of total lumbar disc replacement with SB Charité III prosthesis.

METHODSFrom Dec 1999 to Dec 2006, total lumbar disc replacement with SB Charité III prosthesis was performed in 65 patients affected with degenerative lumbar disc disorders. Among these patients, 48 (52 prosthesis) were followed up for more than two years (from 2.0 to 7.5 years). There were 22 males and 26 females with an average age of 43 years old (from 36 to 58 years). The diagnosis was lumbar disc herniation with low back pain in 34 patients, discogenic low back pain in 9 patients and failed lumbar disc surgery in 5 patients. All patients underwent standard anterior procedure under general anesthesia. One level replacement was done in 44 patients (L3,4 in 3, L4,5 in 23 and L5-S1 in 18), and two level procedures in 4 patients (L3,4/L4,5 in 1 and L4,5/L5-S1 in 3). Clinical and radiographic results of these patients were evaluated at each follow-up time (1, 3, 6, 12, 24 months after operation and the latest).

RESULTSThe average visual analogue scales score for pain was 9.3 before operation, changed to 4.3 one month after operation, further declined to 2.6 two years after operation and finally to 1.8 at the latest follow-up evaluation. Meanwhile, the average Oswestry Disability Index was 45.8 before operation, 28.6 one month after operation, 12.5 two years after operation and 8.2 at the latest followup evaluation. All operated levels but one maintained mobile and there was no significant loss of range of motion observed. Complications such as implant dislocation or significant subsidence of the prosthesis occurred in none case of this group. All patients but one (98%) were satisfied with the surgery at the latest follow-up evaluation.

CONCLUSIONSTotal lumbar disc replacement is an effective method for the treatment of degenerative disc disorders. Its long-term outcome remains to be verified.

Adult ; Arthroplasty, Replacement ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc ; surgery ; Joint Prosthesis ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

Objective To determine the expression of cathepsin L2 (CTSL2)and evaluate its activity in skin lesions of seborrheic keratosis (SK),to observe the ultrastructural changes of melanosomes in the skin lesions of SK,and to estimate the effect of CTSL2 on the degradation of melanosomes.Methods Twenty patients with SK were enrolled from the Department of Dermatology,Renmin Hospital of Wuhan University.The lesional tissue and the perilesional normal skin were biopsied from each patient.Among 15 of the 20 patients,hematoxylin and eosin (HE)staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules,transmission electron microscopy (TEM)was conducted to observe the ultrastructural changes of melanosomes,and immunohistochemical staining was performed to estimate the cellular proliferative activity.RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity,respectively.Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent.The purified melanosomes were incubated with epidermal lysates of SK lesions,and TEM was used to observe the changes in the membrane structure of melanosomes.Statistical analysis was carried out by paired t test,and a P value ＜ 0.05 was considered statistically significant.Results A large number of melanin granules were deposited in SK lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00％ ± 4.00％) than in the SK lesions (24.33％ ± 3.06％)(t =8.49,P ＜ 0.05).RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA:0.35 ± 0.09 vs.0.43 ± 0.08,t =3.17,P ＜ 0.05;activity:17.46 ± 0.45 vs.28.78 ± 0.58,t =34.29,P ＜ 0.05).Moreover,TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33％ ± 4.93％) than in the normal skin lysate-treated group (43.00％ ± 2.65％,t =3.30,P ＜ 0.05).Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes.However,whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.