The genes of short armed hammerhead ribozyme targeting against two sites on positive strand (194-196) and negative strand (89-91) of ASSVd were designed, synthesized and cloned according to the action manner of hammerhead ribozyme. The full lengths of the genes are 42 bp (RzASSVd(+)) and 40 bp (RzASSVd(-)). After transcription in vitro, the ASSVd positive and negative RNA labeled with 32P were mixed with the ribozyme transcript and incubated 3-4 h at 50 degrees C or 37 degrees C. The results were assayed on 8% PAGE (containing 8 mol/L urea) and autoradiogrammed. As predicted, the transcript of the active RzASSVd(-) could cleave the ASSVd negative strand RNA with a high activity but had no cleavage effect on the ASSVd positive strand. The transcript of the RzASSVd(+) gene could cleave the ASSVd positive strand but its cleavage activity was very low. As the same, it cannot cleave the negative strand either. On the base of the result, we construct dimmer ribozyme gene pGEMRzASSVd(+/-) containing both RzASSVd(+) and RzASSVd(-).
Cloning, Molecular ; Malus ; virology ; Plant Diseases ; virology ; RNA, Catalytic ; genetics ; metabolism ; therapeutic use ; RNA, Viral ; metabolism ; Substrate Specificity ; Transcription, Genetic ; Viroids ; metabolism

A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3' end of hammerhead ribozyme was synthesized, amplified and cloned at the Xho I-Hind III site of pGEM7Zf(+). The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2, 4, 6, 8, 10 and 12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1:1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.
Malus ; virology ; RNA, Catalytic ; chemistry ; genetics ; metabolism ; RNA, Viral ; metabolism ; Viroids ; metabolism

OBJECTIVETo observe the effect of 25-hydroxyvitamin D3 on the permeability and ZO-1 expression in normal human airway epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-hour exposure to different concentrations of 25-hydroxy vitamin D3, and the transepithelial electrical resistance (TER) of the cell monolayer was measured using a Millicell-ERS voltohmmeter. Real-time quantitative RT-PCR was employed to determine the changes of ZO-1 mRNA expression in the cells following the exposures.

RESULTSExposure to 25-hydroxyvitamin D3 resulted in significantly increased permeability of 16HBE cells, but the exspression of ZO-1 showed no obvious changes. 25-hydroxyvitamin D3 at 4×10(-9) mol/L showed the strongest effect in increasing the permeability of cell monolayer.

CONCLUSION25-hydroxyvitamin D3 increases the permeability of normal bronchial airway epithelial cell monolayer in vitro, but this effect is not mediated by upregulation of ZO-1 expression.

Bronchi ; cytology ; metabolism ; Calcifediol ; pharmacokinetics ; pharmacology ; Cell Line ; Cell Membrane Permeability ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Zonula Occludens-1 Protein ; genetics ; metabolism

In this paper we introduce a new algorithm about ST-segment--Triangle way. At the same time, based on analyzing all kinds of algorithms, we have summarized a compositive algorithm used to detect the emulating data. The testing result shows that the algorithm, which is easy to use, has a high speed and a good accuracy.
Algorithms ; Electrocardiography ; methods

AIMTo investigate the effect of interleukin-8 (IL-8) on the differentiation and clonal expansion of 3T3-L1 preadipocyte during the differentiation period.

METHODSThe morphological changes of 3T3-L1 cells during differentiation after the treatment of IL-8 was observed by Oil-Red O staining. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured by a spectrophotometric method. MTT method and 3H-TdR incorporation were applied to examine the changes of cell proliferation and DNA synthesis in clonal expansion of 3T3-L1 cells. Cell cycle analysis was taken by flow cytometry.

RESULTSIL-8 could inhibit the differentiation and GDPH activity in a dose dependent manner. IL-8 decreased the cell proliferation and DNA synthesis in clonal expansion after induction. Also, the proportion of cells in G1 phase was increased and that of cells in S and G2 phase was declined after the treatment of IL-8.

CONCLUSIONIL-8 inhibits the differentiation of 3T3-L1 preadipocytes by decreasing the clonal expansion of the cells.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; drug effects ; Interleukin-8 ; pharmacology ; Mice

Objective To assess the clinical effects of mouse nerve growth factor (NGF) on prevention of cerebral trauma-induced dysfunction. Methods Thirty-three patients with cerebral trauma, admitted to our hospital from Mar. 2007 to Feb. 2009, were chosen as NGF-treated group and NGF were injected intramuscularly at a daily dose of 40 μg. Control group was composed of another 33 patients, whose consciousness and traumatic condition were comparable with those of NGF-treated group and conventional treatment was given. The data of Glasgow coma scale (GCS) and Karnofsky performance status (KPS) were analyzed in the 2 groups. Results The NGF-treated group showed significantly high CCS 14 d after treatment as compared with the control group (P<0.05). The KPS in the NGF-treated group was statistically higher than that in the control group 6 months after the treatment was ended (P<0.05). Conclusion NGF, one of the most important neurotrophin, is safe and effective in the prevention of postinjury dysfunction.

OBJECTIVETo evaluate the effect of insular pectoralis major myocutaneous flap on repair of jaw and neck tissue defect as a result of high-voltage electric burn.

METHODSEighteen patients with large area tissue defect in jaw and neck caused by high-voltage electric burn hospitalized from August 2001 to December 2007 were repaired with insular pectoralis major myocutaneous flap. The flaps (from 12 cm x 10 cm to 16 cm x 13 cm) was transplanted in 8 patients after early wound debridement, and in 10 patients on infected wounds.

RESULTSAll flaps survived wells except in 4 patients necrotic area (length 2-5 cm, width 1-2 cm) appeared in the distal wound edge, localized dehiscence of wound edge in 2 cases, which were healed after dressing change or secondary suturing. Patients were followed up from 6 months to 3 years, and the appearance and function were satisfactory.

CONCLUSIONSPectoralis major muscle island myocutaneous flap has the advantage of large tissue mass, with thin and flat muscle belly, and rich blood supply, which is suitable for repair of large deep tissue defect in jaw and neck.

Adolescent ; Adult ; Burns, Electric ; surgery ; Female ; Humans ; Middle Aged ; Muscle, Skeletal ; transplantation ; Neck ; surgery ; Skin Transplantation ; Surgical Flaps ; Young Adult

OBJECTIVETo summarize the experience in the treatment of severe pressure sore.

METHODSFrom Aug. 2007 to Jun. 2011, 21 cases of severe pressure sore with 43 III-IV degree lesions were treated with combination treatment, including vacuum sealing drainage technique, local fascia flaps, upper or lower gluteus maximus island myocutaneous flaps, lower gluteus maximus myocutaneous flap, neurocutaneous femoris posterior flaps, tensor fascia lata island myocutaneous flaps, free latissimus dorsi myocutaneous flaps, and skin graft, combined with stryker frame and nursing tracking guidance. 13 of 21 cases had multiple pressure sore. Among them, 5 III degree pressure sores were covered by skin grafting and 3 non-caudal III degree pressure sores (< 2 cm in width) were directly closed after debridement. 8 of 21 cases had single IV degree pressure sore.

RESULTSAll the 43 wounds healed completely. 5 wounds in 3 cases had effusion under flap which healed after re-drainage. The wounds were not healed in 3 cases with flap transposition which were also healed after re-debridement. All the flaps survived completely. 16 cases were followed up for 2-26 months. Recurrence happened in 4 cases after discharge because of not following the required nursing care.

CONCLUSIONSComprehensive application of vacuum sealing drainage technique, multiple myocutaneous flaps and skin grafting, combined with stryker frame and nursing tracking guidance after discharge can be used for the treatment of severe pressure sore with satisfactory results.

Adolescent ; Adult ; Aged ; Combined Modality Therapy ; Debridement ; Drainage ; Female ; Humans ; Male ; Middle Aged ; Pressure Ulcer ; surgery ; therapy ; Skin Transplantation ; Surgical Flaps ; Treatment Outcome ; Young Adult

Total aralosides of Aralia elata (Miq) Seem (TASAES) from Chinese traditional herb Longya Aralia chinensis L was found to improve cardiac function. The present study was to determine the protective effects of TASAES on diabetic cardiomyopathy, and the possible mechanisms. Therefore, a single dose of streptozotocin was used to induce diabetes in Wister rats. Diabetic rats were immediately treated with low, medium and high doses of TASAES at 4.9, 9.8 mg/kg and 19.6 mg/kg body weight by gavage, respectively, for eight weeks. Cardiac function was evaluated by in situ hemodynamic measurements, and patch clamp for the L-type Ca2+ channel current (ICa2+-L) and transient outward K+ channel current (Ito). Histopathological changes were observed under light and electron microscope. The expression of pro-fibrotic factor, connective tissue growth factor (CTGF) was monitored using immunohistochemistry staining. Compared with diabetic group, medium and high doses, but not low dose, of TASAES showed a significant protection against diabetes-induced cardiac dysfunction, shown by increased absolute value of left ventricular systolic pressure (LVSP) and maximum rates of pressure development (+/-dp/dt(max)), and enhanced amplitude of ICa2+-L (P < 0.05). Histological staining indicated a significant inhibition of diabetes-caused pathological changes and up-regulation of CTGF expression (P < 0.05). The results suggest that TASAES prevents diabetes-induced cardiac dysfunction and pathological damage through up-regulating ICa2+-L in cardiac cells and decreasing CTGF expression.
Animals ; Aralia/*chemistry ; Calcium Channels, L-Type/physiology ; Cardiomyopathies/*drug therapy/etiology/physiopathology ; Connective Tissue Growth Factor/metabolism ; Diabetes Mellitus, Experimental/*complications ; Drugs, Chinese Herbal/*chemistry ; Heart/drug effects/physiopathology ; Hemodynamics ; Male ; Myocardium/pathology ; *Oleanolic Acid/analogs & derivatives/therapeutic use ; Patch-Clamp Techniques ; Potassium Channels/physiology ; Rats ; Rats, Wistar ; Saponins/*therapeutic use ; Treatment Outcome

OBJECTIVETo study the abnormal reactions of a series of free radicals and the oxidative damages induced by free radical abnormal reactions in the bodies of patients with chronic glomerulonephritis.

METHODSEighty chronic glomerulonephritis patients (CGNP) and eighty healthy adult volunteers (HAV) were enrolled in a random control study, in which concentrations of nitric oxide (NO) in plasma, lipoperoxides (LPO) in plasma and in erythrocytes, and vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in erythrocytes were determined with spectrophotometric assays.

RESULTSCompared with the average values of the above biochemical parameters in the HAV group, the average values of NO in plasma, and LPO in plasma and erythrocytes in the CGNP group were significantly increased (P = 0.0001), while those of VC, VE and beta-CAR in plasma as well as those of SOD, CAT and GPX in erythrocytes in the CGNP group were significantly decreased (P = 0.0001). Pearson product-moment correlation analysis showed that with increase of the concentration of blood creatinine as well as prolongation of the course of disease in the CGNP, the concentrations of NO in plasma, and LPO in plasma and erythrocytes in the CGNP increased gradually, while the concentrations of VC, VE and beta-CAR in plasma as well as the activities of SOD, CAT and GPX in erythrocytes in the CGNP decreased gradually (P = 0.002454-0.000001). The relative risk ratio (RR) of the above biochemical parameters reflecting oxidative damages in the bodies of CGNP ranged from 6.061 to 72.429. The reliability coefficient (alpha) that the above biochemical parameters were used to reflect the oxidative damages of the CGNP was 0.8137, standardized item alpha = 0.9728, Hotelling's T-Squared = 1135680.191, F = 53274.6478, P = 0.000001.

CONCLUSIONSThe findings in this study show that in the bodies of CGNP a series of free radical chain reactions result in severe pathological aggravation and induce oxidative damages in their bodies. Therefore, suitable dose of antioxidants should be supplemented to them so as to alleviate oxidative damages in their bodies.

Adult ; Antioxidants ; pharmacology ; therapeutic use ; Catalase ; pharmacology ; Chronic Disease ; Erythrocytes ; enzymology ; Female ; Free Radicals ; adverse effects ; Glomerulonephritis ; physiopathology ; Glutathione Peroxidase ; pharmacology ; Humans ; Lipid Peroxidation ; Male ; Nitric Oxide ; adverse effects ; analysis ; Oxidation-Reduction ; Oxidative Stress ; Superoxide Dismutase ; pharmacology