OBJECTIVETo investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell (HUVECs) and the effects on hydrogen-peroxide (H2O2)-induced oxidative stress injury in these cells.

METHODSWith the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni2+-NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 (0.5 mmol/L) alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents were measured.

RESULTSThe PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), while LDH leakage (849.3 U/L+/-95.1 U/L) and MDA (8.23 nmol/L+/-1.58 nmol/L) content were significantly higher than that in control group (540.6 U/L+/-65.7 U/L and 2.46 nmol/L+/-1.42 nmol/L, respectively, all P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury.

CONCLUSIONThe PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.

Catalase ; metabolism ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Hydrogen Peroxide ; Oxidative Stress ; physiology ; Peptides ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo study the effect and the mechanism of Xuesaitong drop pills (total saponins in Radix Notoginseng; XDP) on experimental thrombosis, thrombolysis and blood theology.

METHODFirst, the rats were randomly divided into five groups: control, XDP (90, 30, 10 mg x kg(-1)), Xuesaitong tablet (XP) 30 mg x kg(-1). Then the effect of the drugs on thrombus and thrombosis was studied after the ratsthrombosis was induced by the arteriovenous shunt. Second, the rats were randomly divided into seven groups: model, XDP (90, 30, 10 mg x kg(-1)), XT (90, 30 mg x kg(-1)), lumbrukinase capsule. Then the effect of the drugs on thrombus and thrombosis was studied after the rats'thrombosis was induced by the electrical stimulation of common carotid artery. Third, the rats were randomly divided into six groups: control, model, XDP (80, 40 mg x kg(-1)), XT (40, 20 mg x kg(-1)). Then the effect of the drugs on blood circulation promoting was observed after the rats'acute blood stasis induced by adrenalin and icy water.

RESULTXDP 90, 30 mg x kg(-1) could notably lighten the wet-weight and dry-weight of thrombus in the arteriovenous shunt model in rats in a dose-dependent manner (P < 0.01). XDP 90 mg x kg(-1) with intragastric administration for 3 days had the satisfactory effect on thrombolysis after the rat's thrombosis was induced by the electrical stimulation of common carotid artery (P < 0.01). XDP 80, 40 , 20 mg x kg(-1) reduced significantly erythrocyte aggregation (P < 0.01) and decreased the whole blood viscosity at low shear rate (P < 0.05). XDP 80, 40 mg x kg(-1) reduced the whole blood viscosity at high shear rate and plasma viscosity (P < 0.05). XDP 80 mg x kg(-1) decreased the whole blood viscosity at high shear rate (P < 0.05).

CONCLUSIONXDP can significantly inhibit the thrombosis and has the satisfactory effect on thrombolysis. One kind of the mechanism is related to the effect on blood rheology.

Animals ; Blood Viscosity ; drug effects ; Carotid Artery Thrombosis ; blood ; drug therapy ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Erythrocyte Aggregation ; drug effects ; Erythrocyte Deformability ; drug effects ; Female ; Fibrinolytic Agents ; pharmacology ; therapeutic use ; Male ; Panax notoginseng ; chemistry ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Wistar ; Saponins ; isolation & purification ; pharmacology ; therapeutic use ; Thrombolytic Therapy

OBJECTIVETo investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells.

METHODSTwo prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method.

RESULTSEGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L.

CONCLUSIONPEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.

Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Green Fluorescent Proteins ; metabolism ; Humans ; Peptides ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; toxicity ; Umbilical Veins ; cytology

ObjectiveTo introduction of perforator flaps,muscle flaps pedicled by descending branch of lateral circumflex femoral artery,method and their clinical application that multiple soft tissue defects of hand are repaire by muliplefoliated tissue flap only branch lateral circumflex femoral artery.MethodsFifteen patients with multiple soft tissue defects of hand were repaired muliplefoliated tissue flap only pedicled bydescending branch lateral circumflex femoral artery.At first,the anterolateral thigh perforator flap was designed and harvested according to the soft tissue defects of hand, then the descending branch lateral circumflex femoral artery was dissected at the same time the segmented perforator flap,fascia lata flap,rectus femoris muscle flap, vastus lateralis muscle flap, vastus intermedius muscle flap and distal spatium intermusculare flap were harvested in need according to distance among soft tissue defects.The muliplefoliated tissue flap was harvested only pedicled by descending branch lateral circumflex femoral artery, at last muscle flaps and fascia lata flaps were covered by skin graft, so the multiple soft tissue defects of hand were repaired in one time.ResultsNo vascular crisis happened. All skin grafts survived well, the contour of all repaired soft tissue defects was good and protective feeling was recovered by skin grafts of all flaps. All cases were got follow-up and the range was from 6 to 20 months(the average was 8.7 months).Wound of donor site healed well, muscle strength of quadriceps and motion of knee were normal. Three cases were excellent,nine cases were well and 3 cases were good, according to upper extremity function evaluation criteria of Chinese Medical Society for the Surgery of the Hand, the rate of good was 80 percent.ConclusionMultiple soft tissue defects of hand can be repaired by muliplefoliated flap only pedicled by descending branch of lateral circumflex femoral artery. Its advantages included reduction of operation time and treatment, good recovery of hand contour and function. It is a good method to repair multiple soft tissue defects of hand.

OBJECTIVEThe study was to evaluate the method of early laser intervention of hemangioma in facial and neck regions of infant.

METHODSBetween January 1999 and December 2006, twelve patients, aged 6 days to 3 months, with cutaneous hemangioma in facial and neck regions, were treated with laser, eight cases with Nd:YAG laser therapy and four cases with Venus laser therapy. Four cases with hemangioma in facial and neck regions of infant treated with oral corticosteroid were as control. The outcome was recorded with 1 to 6 years of follow-up.

RESULTSTotal resolution was obtained in twelve patients with laser intervention. Atrophic scars occurred in eight patients with Nd:YAG laser therapy, without other complications, such as ulceration, life-threatening hemorrhage and et al. No scar occurred in four patients with Venus laser therapy. Recurrence was not seen in twelve cases with laser therapy with follow-up. Hemangiomas enlarged continuously in four cases with oral corticosteroid therapy.

CONCLUSIONEarly laser intervention is an excellent management of cutaneous hemangioma in facial and neck regions of infant.

Face ; Female ; Head and Neck Neoplasms ; Hemangioma ; Humans ; Infant ; Laser Therapy ; Lasers ; Light ; Male ; Neck

Fifteen known compounds were isolated from Swertia delavayi by silica gel, Sephadex LH-20 and Rp-18 column chromatographies. Based on extensive spectroscopic analysis (MS, 1H, 13C-NMR), their structures were identified aserythrocentaurin (1), erythrocentaurindimethylacetal (2), sweroside (3), swertiamarin (4), gentiopicroside (5), swertiakoside A (6), 2'-O-acetylswertiamarin (7), 4'-O-[(Z) -coumaroyl] swertiamarin (8), 1,5,8-trihydroxy-3-methoxyxanthone (9), 8-O-β-D-glucopyranosyl-1-hydroxy-2,3, 5-trimethoxyxanthone (10), 8-O-[β-D-xyl- opyranosyl-(1 --> 6)-β-D-glucopyranosyl]-7,8-dihydroxy-3-methoxyxanthone (11), isovitexin (12), β-sitosterol (13), daucosterol (14), and oleanolic acid (15). Among them, ten ones (14, 7-11, 13) were obtained from S. delavayi for the first time. The isolates were evaluated for their anti-HBV activities in HepG 2. 2. 15 cell line in vitro. The results showed that compound 1, 2, 6, 7, 9 and 12 exhibited significant inhibitory activity on HBV DNA replication with IC50 values from 0.05 to 1.46 mmol x L(-1).
Antiviral Agents ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Hepatitis B virus ; drug effects ; genetics ; Magnetic Resonance Imaging ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Swertia ; chemistry

OBJECTIVETo construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT.

METHODSUsing pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro.

RESULTSSequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g.

CONCLUSIONThe prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress.

Base Sequence ; Blotting, Western ; Catalase ; genetics ; metabolism ; Chromatography, Affinity ; Cloning, Molecular ; Cysteamine ; analogs & derivatives ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Molecular Sequence Data ; Peptides ; genetics ; metabolism ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1.

METHODSTwo oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) was purified with Ni(2+)-resin affinity chromatography and transduced into human umbilical vein endothelial cells.

RESULTSSequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells.

CONCLUSIONThe successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.

Base Sequence ; Blotting, Western ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Endothelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Molecular Sequence Data ; Peptides ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Umbilical Veins ; cytology

Objective To discuss the effects of different exercise frequency and diet guidance for fat loss on obese patients, and to develop a safe and effective multidisciplinary intervention program for obese patients. Methods A total of 140 members of Ningbo Slimming Club were recruited, with the age between 18-60 years, body mass index (BMI) greater than 28 kg/m2, and they were randomly assigned to low frequency group and high frequency group. In low frequency group, the intervention was complete 100 minutes aerobic exercise and 3 times a week. In high frequency group, the intervention was complete 50 minutes of aerobic exercise and 6 times a week. Each group followed the same diet weight loss program. By comparing each index before and after the intervention, the effect of fat loss was evaluated. Results After intervention, systolic blood pressure, diastolic blood pressure, body weight, BMI, waist circumference, body fat percentage, triglyceride, low density lipoprotein and total cholesterol in two groups were decreased, and high density lipoprotein were increased (P<0.05) . Compared between the low frequency and high frequency exercise group, there were no significant difference for the systolic blood pressure, diastolic blood pressure, body weight, waist circumference, BMI, triglyceride, high density lipoprotein, low density lipoprotein and total cholesterol levels (P>0.05), but body fat percentage in low frequency exercise group decreased significantly (P< 0.05) . Conclusion Diet intervention combined with exercise has a significant effect of fat loss on obese patients. Based on the same weekly aerobic exercise time, obese patients with low frequency of exercise had better fat loss than obese patients with high frequency of exercise.

OBJECTIVE: To investigate the mechanism of losartan treating glomerulosclerosis and to observe the effect of losartan on the expressions of TGF-beta1, p-Smad2/3, and Smad7 in the renal tissues of 5/6 nephrectomized rats. METHODS: Male Wistar rats were randomly divided into a sham-operated group, a 5/6 nephrectomized model group, and a losartan treated group. The rats in the model group and the losartan treated group were performed 5/6 nephrectomy by the method with 2 procedures. Twelve weeks after of the operation, all rats were killed. The 24-hour urinary protein, serum creatinine, and urea nitrogen were detected. Pathological changes of the renal tissues were observed by HE and Masson staining, and the expressions of TGF-beta1, p-Smad2/3, and Smad7 were detected by immunohistochemical staining. RESULTS: The 24-hour urinary protein, serum creatinine, urea nitrogen, and the relative area of collagen in the renal tissues of the rats in the model group significantly increased (P<0.01), and losartan could reduce these indexes. The expressions of TGF-beta1 and p-Smad2/3 were just at a low level in the renal tissues of the rats in the sham-operated group, and were strongly positive in the model group; but losartan could decrease the expressions of TGF-beta1 and p-Smad2/3 (P<0.01). The expression of Smad7 in the model group was fewer than that in the sham-operated group (P<0.01), but losartan could improve the expression of Smad7 (P<0.01). CONCLUSION Losartan may implement its anti-glomerulosclerosis by affecting TGF-beta1, p-Smad2/3, and Smad7 of TGF-beta/Smads pathway of the renal tissues of 5/6 nephrectomized rats.
Animals ; Kidney ; drug effects ; metabolism ; Losartan ; pharmacology ; Male ; Nephrectomy ; methods ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism