An efficient protocol for plant regeneration from protoplasts of the methionine resistant variant of Astragalus melilotoides was established. The friable calli induced from internode segments of variant plants were used for protoplast preparation. The protoplasts were isolated through enzyme digestion. Calli were formed after sustained divisions of protoplasts. High frequency of shoot differentiation was obtained from the protocalli on differentiated medium. The effects of different media, culturing methods and plating densities on protoplast divisions and plant regeneration were studied. The results show that agarose-beads culture method, KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5mg/L 6BA, 0.3 mol/L mannitol, 2% (W/V) sucrose and 500 mg/L casein hydrolysate at a plating density of 3 x 10(5)/mL are the appropriate conditions for protoplast division of the methionine resistant cell line. The division frequency is over 38%. The protoplast-regenerated plants still preserve resistance to methionine and ethionine.This research builds up the foundation for the resistant cell line as a parent of somatic hybridization.
Astragalus Plant ; growth & development ; physiology ; Culture Media ; Culture Techniques ; methods ; Drug Resistance ; Methionine ; pharmacology ; Protoplasts ; cytology ; Regeneration

Multi-component traditional Chinese medicines are an innovative research mode for traditional Chinese medicines. Currently, there are many design methods for developing multi-component traditional Chinese medicines, but their common feature is the lack of effective connection of the traditional Chinese medicine theory. In this paper, the authors discussed the multi-component traditional Chinese medicine design methods based on medicinal property combination modes, provided the combination methods with the characteristics of traditional Chinese medicine for the prescription combinations, and proved its feasibly with hypertension cases.
Animals ; Antihypertensive Agents ; administration & dosage ; chemistry ; Blood Pressure ; drug effects ; Drug Combinations ; Drug Therapy ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Humans ; Hypertension ; drug therapy ; physiopathology ; Medicine, Chinese Traditional ; Rats

Objective To explore protective effect of Heme oxygenase-1(HO-1) on irradiation-induced endothelial cell apoptosis.Methods Human endothelial cell line EA.hy926 were administered with or without HO-1 activator Copp and/or HO-1 inhibitor Znpp,respectively.Then,cells were treated with or without 8 Gy radiation.The HO-1 protein expression of cells were assessed with Western blotting and apoptosis of cells treated with irradiation were evaluated with flow cytometry.Moreover,cytochrome C releasing into cytosol were also determined by Western blotting. Results In PBS+R group,HO-1 protein expression of EA.hy926 was low posterior to irradiation.When cells were preconditioned with Copp and/or Znpp,then recieved with 8Gy irradiation,the HO-1 protein expression of EA.hy926 increased significantly in comparision with the PBS+R group(P

An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch (Coronilla varia L.) by infecting the segments of cotyledons and hypocotyls of 15d-old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormone-free Murashige and Skoog (MS) medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency (87.4%) was achieved by preculturing cotyledons for 2d and pre-treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase (OD600 = 0.8). The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4-D, 0.5mg/L NAA and 0.5mg/L KT. Globular-, heart-, torpedo-, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.5mg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non-transformed plants. However, the contents of 3-nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR-DNA. PCR analysis confirmed that the TL-DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants.
Fabaceae ; genetics ; growth & development ; physiology ; Plant Roots ; genetics ; growth & development ; physiology ; Plants, Genetically Modified ; genetics ; growth & development ; Regeneration ; Rhizobium ; genetics ; Tissue Culture Techniques ; Transformation, Genetic

OBJECTIVETo construct strains containing green fluorescent protein (GFP) to study gene regulation in Saccharomyces albicans cells during the infection process.

METHODSpACT1-GFP was constructed, and Saccharomyces albicans CAI4 was transformed. The expression of GFP in yeast and hyphal compartments was observed with microscopy.

RESULTS99% of Saccharomyces albicans cells containing pACT1-GFP fusion displayed significant fluorescence levels both in the yeast and hyphal compartments. The fluorescence intensity in two compartments had no obvious difference.

CONCLUSIONpACT1-GFP can be expressed stably in the yeast cells.

Candida albicans ; Green Fluorescent Proteins ; Humans ; Plasmids ; Saccharomyces ; Saccharomyces cerevisiae

Objective To explore the value of Multi-detector CT in spinal cord angiography. Methods Ten patients with initial MR and clinical findings suggestive of spinal cord vessel disease were performed CT spinal cord angiography.Among these,7 patients were performed DSA later within 1 week, and 4 patients were therapy by operation.CT protocol:Toshiba Aquilion 64 slice CT scanner,0.5 mm thickness,0.5/r,120 kV,350 mA,choose aortic arch level as inspection position,and use"surestart" technique with CT threshold 180 HU.Contrast medium was Iohexol(370 mg I/ml),with injection velocity of 6 ml/s.The total volume was 80 ml.The CT spinal cord angiography images were analyzed according to disease model,disease range,feeding artery,fistula,draining veins,and were compared with DSA and operation results.Results All CT spinal cord angiography images displayed spinal vessel malformation. Among these,3 patients were inner-medullary arteriovenous malformation;2 patients were peri-medullary arteriovenous fistula;5 patients were spinal dural arteriovenous fistula.All cases showed disease range,and draining veins clearly,one patient had two vessels that were false positive,and all the other cases showed feeding arteries clearly,which were confirmed by DSA.Conclusion There are great values for CT spinal angiography in diagnosing spinal vessel disease,it can be a screening exam before DSA.

OBJECTIVETo investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3.

METHODSThe effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting.

RESULTSSorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates: sorafenib: 46.16% +/- 2.52%, 5-FU: 28.67% +/- 6.16%, and sorafenib + 5-FU: 22.59% +/- 6.89%. The sorafenib + 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 +/- 27.84) mg/L to (178.61 +/- 20.73) mg/L (P = 0.003). Sorafenib alone induced G1 phase arrest (increasing from 44.73% +/- 1.63% to 65.80% +/- 0.56%; P less than 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63% +/- 0.65% to 22.83% +/- 1.75%; P less than 0.01), as well as down-regulated cyclinD1 expression (0.57 +/- 0.03-fold change vs. untreated control group; P less than 0.01). 5-FU alone up-regulated cyclinD1 expression (1.45 +/- 0.12-fold change vs. untreated control group; P less than 0.01). Moreover, sorafenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways, with the fold-changes of p-C-RAF, p-ERK1/2 and p-STAT3 being 0.56 +/- 0.05, 0.54 +/- 0.02 and 0.36 +/- 0.02, respectively (all P less than 0.01); 5-FU alone produced no significant effects on these pathways.

CONCLUSIONAdministered alone, both sorafenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells. The sorafenib + 5-FU combination treatment produces antagonistic, rather than synergistic, effects. Sorafenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G1phase arrest and S phase reduction, thereby reducing the cells' sensitivity to 5-FU.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Drug Antagonism ; Fluorouracil ; pharmacology ; Humans ; Niacinamide ; analogs & derivatives ; pharmacology ; Phenylurea Compounds ; pharmacology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

In order to study the chemical constituents in the water extract of the stem of Nauclea officinalis, column chromatography over D101 macroporous resin and silica gel and an automatic purification system were used to isolate and purify the chemical constituents from the extract. Nine compounds were obtained. By analysis of the physicochemical properties and spectral data, their structures were identified as naucleamide G (1), 3, 4-dimethoxyphenol-beta-D-apiofuranosyl (1-->6)-beta-D-glucopyranoside (2), kelampayoside A (3), 3alpha, 5alpha-tetrahydrodeoxycordifoline lactam (4), naucleamide A-10-O-beta-D-glucopyranoside (5), pumiloside (6), 3-epi-pumiloside (7), strictosamide (8) and vincosamide (9), separately. Among them, compound 1 is a new compound, compound 2 was found in plants of the genus Nauclea for the first time, and compounds 3 and 4 were isolated from this plant for the first time.
Camptothecin ; analogs & derivatives ; chemistry ; isolation & purification ; Carbolines ; chemistry ; isolation & purification ; Glucosides ; chemistry ; isolation & purification ; Indole Alkaloids ; chemistry ; isolation & purification ; Molecular Structure ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry ; Rubiaceae ; chemistry ; Vinca Alkaloids ; chemistry ; isolation & purification

Objective: To observe the clinical efficacy of auricular point pricking-bloodletting plus auricular point sticking therapy for acne vulgaris. Methods: A total of 66 patients with acne vulgaris were randomized into an observation group and a control group by the random number table, with 33 cases in each group. The observation group was treated with auricular point pricking-bloodletting plus auricular point sticking therapy, and the control group was treated only with auricular point sticking therapy. The treatments of both groups were performed twice a week, 4 weeks as a course of treatment, for 3 courses in total. The scores of skin lesions and dermatology life quality index (DLQI) scores were recorded before and after treatment to assess the clinical efficacy. Results: During the trial, there were 3 cases of drop-out both in the observation group and the control group. After 3 courses of treatment, the total effective rate of the observation group was 96.7％, while that of the control group was 76.7％. The difference between the two groups was statistically significant (P<0.05). The intra-group comparison showed that the scores of skin lesion and DLQI were both decreased with the increase of treatment times, that was, the scores were lower than those at the previous time point (allP<0.05). After 1, 2, and 3 courses of treatment, the scores of skin lesion and DLQI of both groups were statistically different from those of the same group before treatment (allP<0.05). At every time point during the treatment, the scores of skin lesion and DLQI of the observation group were lower than those of the control group, and the differences between the two groups were statistically significant (all P<0.05). Conclusion: Auricular point pricking-bloodletting plus auricular point sticking has a better curative effect than auricular point sticking therapy alone in the treatment of acne vulgaris, and has a time-effect correlation.

This study was to investigate the changes of autonomic nerve function and hemodynamics in patients with vasovagal syncope (VVS) during head-up tilt-table testing (HUT).HUT was performed in 68 patients with unexplained syncope and 18 healthy subjects served as control group.According to whether bradycardia,hypotension or both took place during the onset of syncope,the patients were divided during the test into three subgroups:vasodepressor syncope (VD),cardioinhibitory syncope (CI) and mixed syncope (MX) subgroups.Heart rate,blood pressure,heart rate variability (HRV),and deceleration capacity (DC) were continuously analyzed during HUT.For all the subjects with positive responses,the normalized low frequency (LFn) and the LF/HF ratio markedly decreased whereas normalized high frequency (HFn) increased when syncope occurred.Syncopal period also caused more significant increase in the power of the DC in positive groups.These changes were more exaggerated compared to controls.All the patients were indicative of a sympathetic surge in the presence of withdrawal vagal activity before syncope and a sympathetic inhibition with a vagal predominance at the syncopal stage by the frequency-domain analysis of HRV.With the measurements ofDC,a decreased vagal tone before syncope stage and a vagal activation at the syncopal stage were observed.The vagal tone was higher in subjects showing cardioinhibitory responses at the syncopal stage.DC may provide an alternative method to understand the autonomic profile of VVS patients.