Adult ; Diagnostic Errors ; Humans ; Leprosy, Lepromatous ; diagnosis ; Male ; Sinusitis ; diagnosis

OBJECTIVETo explore the effect of Shenxiong Huayu Capsule (SHC) on the cerebral ischemia-reperfusion (IR) injury and the expression of growth associated protein 43 (GAP43) after total cerebral IR in the hippocampal CA1 region of rats.

METHODSTotally 100 male adult SD rats were randomly divided into five groups, i.e., the control group, the model group, the group A (by taking SHC once daily), the group B (by taking SHC twice daily), and the group C (by taking SHC thrice daily), 20 in each group. The total IR model was prepared by improved Pulsinelli's 4-vessel occlusion method. Morphological changes of the hippocampal CA1 region were observed by HE staining at day 1, 3, 7, and 14. The expression of GAP43 in the hippocampal CA1 region was detected using immunohistochemical assay at day 1, 3, 7, and 14. Meanwhile, the behavioral score was determined. The expression of GAP43 in the hippocampal CA1 region was detected using Western blot at day 14.

RESULTSCompared with the control group, the expression of GAP43 increased in the model group, the behavioral score was elevated, degenerated neurons increased, and survival neurons decreased in the model group (all P < 0.05). Compared with the model group, the expression of GAP-43 increased (with the most significant difference seen in the group C, P < 0.01), the behavioral score significantly decreased, degenerated neurons decreased, and survival neurons increased in each HSC group (all P < 0.05). Survival neurons obviously increased at day 14, of which, most number of survival neurons and highest contents of GAP43 protein could be seen in the group C, showing statistical difference when compared with those of the group A and the group B (P < 0.01).

CONCLUSIONSHC had protective effect on total cerebral IR in the hippocampal CA1, which might be associated with increased expression of GAP43.

Animals ; Brain Ischemia ; metabolism ; CA1 Region, Hippocampal ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; GAP-43 Protein ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

BACKGROUND:Lipoic acid, with a closed circle structure composed by sulphur and carbon atoms, exerts strong anti-oxidation, and has been extensively applied in the prevention and treatment of oxidative stress, diabetic cataract, diabetic neuropathy and cardiovascular diseases. OBJECTIVE:To investigate the protective effect of lipoic acid on peripheral nerve function during peripheral nerve ischemia/reperfusion injury. METHODS:Models of peripheral nerve ischemia/reperfusion injury were established in rabbits, and then rabbit models were then allotted to treatment and non-treatment groups. The treatment group was subdivided into experimental (injection of lippoic acid) and control groups according to the use of lipoic acid at 1, 3 and 6 hours after ischemia and before reperfusion. The ultrastructural changes of the sciatic nerve were observed under electron microscope, and the electrophysiological changes of the sciatic nerve were detected using evoked potential instrument. RESULTS AND CONCLUSION:With the ischemic time increasing, the number of vacuoles in the axon increased gradually, accompanied by axonal atrophy, and Waller's degeneration in the aggregated microfilaments. The myelin sheath thickening and dissolving were visible. All above phenomena became severest at 6 hours after ischemia. Compared with the control groups, lipoic acid reduced the number of the vacuoles in the axon and all eviated axonal atrophy, Waller's degeneration and demyelination. As the ischemic time increasing, the latency of sciatic nerve was significantly increased, and peaked at 6 hours of ischemia;while the amplitude was significantly decreased, and reached a minimum at 6 hours of ischemia. Compared with the control groups, in the experimental groups, the latency of sciatic nerve was significantly decreased, but the amplitude was significantly increased. These results suggest that lipoic acid provides neuroprotection against peripheral nerve ischemia/reperfusion injury.

Fourteen compounds were isolated from 95% ethanol extract by silica gel, MCI, and ODS column chromatography. These compounds were respectively identified as quercetin (1), kaempferol (2), (+)-catechin (3), fraxin (4), protocatechuic acid (5), gallic acid (6), methyl gallate (7), ethyl gallate (8), apocynol A (9), baccatin (10), cerevisterol (11), ellagic acid (12), 3, 3',4'-tri-0-methylellagic acid(13) and N-benzoyl-L-phenylalaninyl-N-benzoyl-L-phenylalaninate(14) by analyzing their spectral data and comparing with the previously reported literatures. Except for gallic acid (6), all other compounds were isolated from this plant for the first time. Compounds 1, 2 and 6 showed moderate anti-proliferation activities on tumor cells.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Euphorbiaceae ; chemistry ; Humans ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Electrospray Ionization

Objective To analyze the relationships between the drinking water fluoride and bone mineral density (BMD), and serum osteocalcin (BGP) and to explore the BMD and serum BGP as significant early screening biomarkers for fluorosis especially for early bone damage in endemic fluorosis areas. Methods Wamiao (severe endemic fluorosis area, as fluoride exposed group) and Xinhuai (non endemic fluorosis area, as control group) Village were selected in 2006. One hundred and fouty-six objects were chosen from 2 villages (103 in Wamiao, 43 in Xinhuai). The sex, age, body height, body weight, drinking water fluoride in each object's household well, BMD, and serum BGP were investigated, and the dose-response relationships were analyzed between the drinking water fluoride and BMD, and serum BGP. CurveExpert 1.3 Software was used to fit the dose-response relationships between the rate of abnormal BMD, the rate of abnormal serum BGP, and the drinking water fluoride. Results The levels of drinking water fluoride in males' and females' families in fluoride exposed group were [(2.38±0.68), (2.62±0.91 )mg/L] significant higher than that in control group [(0.35±0.08), (0.36±0.07)mg/L], the difference being statistically significant(t values were 14.27 and 11.08,and P<0.01, respectively). BMD in males in fluoride exposed group [(0.78±0.07)g/cm2] was significant lower than that in control group[(0.83±0.08)g/cm2], the difference being statistically significant (t=2.37,P<0.05). Serum BGP in males and females in fluoride exposed group [(4.17±0.67), (4.11±0.57) μg/L] were significant higher than that in control group [(1.48±0.40), (1.44±0.39)μg/L], the difference being statistically significant (t values were 17.64 and 19.40, and P<0.01, respectively]. BMD in the group with drinking water fluoride≥2.92 mg/L[(0.66±0.15 )g/cm2] was significant lower than that in the group with drinking water fluoride<0.42 mg/L [(0.76±0.12)g/cm2], the difference being statistically significant (P<0.01). The levels of serum BGP in the groups with the drinking water 0.42-,2.05-, ≥.92 mg/L[(3.83±1.07), (4.22±0.72), (3.99±0.63) μg/L] were significant higher than that in the group with the drinking water<0.42 mg/L [(1.44±0.37) μg/L], the difference being statistically significant (P<0.01). The equation for the dose-response relationship between the drinking water fluoride and the rate of abnormal BMD was y=(0.284-0.058x)-1.260, r=0.999 94; and y=100.05/(1+78.62e-4.5x), r=0.999 99 for the drinking water fluoride and the rate of abnormal serum BGP. Conclusions There were significant dose-response relationships between drinking water fluoride and BMD and serum BGP. It indicated that BMD and BGP might be considered as early screening biomarkers for endemic fluorosis, especially for the bone damage.

Objective To evaluate the clinical significance of sinus heart rate turbulence(HRT)and the heart rate variability(HRV)in the patients with essential hypertension(EH).Methods HRV and HRT examination were carried out in sixty patients with EH.A subgroup of patients receive metoprolol+nifedipine(n=26)or nife- dipine alone(n=34)were investigated seperately to evaluate the effect of ?-blocker on the HRV and HRT.Fifty healthy persons were served as control.In the HRT determination,turbulence onset(TO)was defined as sinus heart rate acceleration,after ventrieular mature beat while turbulence slope(TS)as sinus heart rate deceleration slope after ventricular premsture beat.Normal value of TO was2.5 ms/RR period. SDNN,RMSSD,LF/HF in HRV were analysed from 24 hours ambulatory electrocardiography(before and after 1 month medication).Results ① In hypertensive group,52 patients showed positive TO(86.6%),48 patients pos- itive TS(80.0%)and 46 patients TO+TS(76.7%),compared with hypertension group only 2(4%)positive TO and 3(6%)positive TS in the healthy control.Forty-two cases SDNN(70.0%),41 cases RMSSD(68.3%)and 38 cases LF/HF(63.3 %)were positive in hypertensive group,while only 1(2 %)SDNN,1(2 %)RMSSD and 3(6 %) LF/HF in control. ② Metoprolol didn't change the positive percentage of parameters in HRT but parameters showing Heart rate variablity in HRV was decreased significantly found between TO,TS and TO+TS.Conclusion HRT and HRV is two indices for determination the dysfunction of autonomic nervous system in hypertension.?-re ceptor blocker inhibit sympathic activity,miligate the decreasing of HRV,but the bipbase accelerative and decelera- tive phenomena of HRT didn't change,HRT seems to be a more sensitive index for monitoring of autonomic sysfunc tion in hypertension.

Purpose To investigate the positive rate and concordance rate of BRAF mutation in papillary thyroid carcinoma detected by real-time PCR method and Sanger sequencing. Methods 312 papillary thyroid carcinomas patients were enrolled in this study. Real-time PCR method and Sanger sequencing were performed to detect BRAF gene mutations. The frequency of BRAF mutation and the concordance of two methods were analyzed. Results BRAF mutation was detected in 65. 4% (204/312) and 63. 8% (199/312) of 312 papillary thyroid carcinoma samples by using real-time PCR method and Sanger sequencing, respectively. There was no significant correlation between BRAF gene mutations and patients’ gender. There was significant correlation between BRAF gene mutations and patients’ age. The overall concordance between real-time PCR method and Sanger sequencing for BRAF mutation detection was 98. 4%. Conclusion Real-time PCR method provides an effective method in BRAF gene mutation detection.

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 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 explore a new approach in gene therapy of ovarian cancer, we used RNAi to inhibit survivin gene expression, and explore the effect of survivin and neu RNAi on growth, apoptosis and chemosensitivity of ovarian cancer cell line SKOV3/DDP cells.

METHODSExpression vector of survivin gene-targeting siRNA was constructed using pSilencer 1.0-U6 vector containing U6 promotor (pSilencer-survivin) and transfected into SKOV3/DDP cells by lipofectamine. The untransfected group and pSilencer-control group were used as control. The expressions of survivin mRNA and protein were identified by RT-PCR and Western blot assay. The proliferation of SKOV3/DDP cells was determined by MTT assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. Cisplatin (DDP) resistance experiment was performed in SKOV3/DDP cells with RNAi.

RESULTSSurvivin RNAi plasmid knocked-down survivin expression in SKOV3/DDP cells obviously, arrested the cells at G(1)/G(0) phase, inhibited the cell proliferation and promoted cell apoptosis. The IC(50) of DDP to SKOV3/DDP cells transfected with survivin siRNA was dropped.

CONCLUSIONSurvivin RNAi can partly suppress the expression of survivin in SKOV3/DDP cells, inhibit the cell proliferation and promote cell apoptosis. Survivin RNAi can enhance the cell sensitivity to apoptosis induced by cisplatin, which implies that survivin RNAi may partly reverse the drug resistance of ovarian cancer. RNAi could be a new approach for gene therapy of cancer.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Inhibitory Concentration 50 ; Microtubule-Associated Proteins ; genetics ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection