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Measuring NO Production by Plant Tissues and Suspension cultured cells

Molecular Plant 2008年第2期1卷 270-284页

作者： Jan Vitecek Vilem Reinohlt Russell L. Jones Department of Plant Biology Faculty of Agronomy Mendel University of Agriculture and Forestry Zemedelska 1 Brno 613 00 Czech Republic Department of Plant and Microbial-Biolocjy University of California Berkeley CA 94720-3102 USA

We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants. The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap. The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors. We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources. NO produced by nitrate reductase （NR） in tobacco leaves and barley aleurone was readily detected, as was the production of NO from nitrite by the incubation medium of barley aleurone. Arabidopsis mutants that overproduce NO or lack NO-synthase （AtNOS1） also displayed the expected NO synthesis phenotype when assayed by our set-up. We could also measure NO production from elicitor-treated suspension cultured cells using this set-up. Further, we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2＇,7＇-difluorofluorescein （DAF-FM）. Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues. In addition to the dramatic effects that pH can have on fluorescence from DAF-FM, the widely used NO scavengers 2-phenyl- 4,4,5, 5-tetramethylimidazoline- 1-oxyl-3-oxide （PTIO） oxide （cPTIO） can produce anomalous and unexpected to bind to dead cells and remain NO-sensitive. and 2-（4-carboxyphenyl）-4,4,5,5-tetramethylimidazoline- 1-oxyl-3- results. Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

关键词： Plant tissue gaseous phase Aleurone Griess reagent cultured cells Tobacco Products NOBELIUM Production

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A new polyphenol, 1, 3-di-O-caffeoyl-5-O-(1-methoxyl-2-O-caffeoyl-4-maloyl)-quinic acid, isolated from cultured cells of Saussurea involucrata

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Chinese Journal of Natural Medicines 2015年第4期13卷 295-298页

作者： ZOU Xiao-Wei LIU Dan LIU Ya-Ping XIU Zhi-Long XIAO Hong-Bin Department of Bioscience and Biotechnology School of Life science and Biotechnology Dalian University of Technology Key Laboratory of Separation Science for Analytical Chemistry Dalian Institute of Chemical Physics Chinese Academy of Sciences Dalian Practical Biotechnology Co. Ltd. Institute of Chinese Materia Medica Chinese Academy of Chinese Medical Sciences

The present study was designed to isolate the polyphenol constituents of cultured cells of Saussurea involucrata. The polyphenol type constituents were isolated using chromatography methods, and then characterized by spectral analysis. 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)-hydrazyl(DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid(ABTS) free radical scavenging were assayed using Vitamin C as the positive control. One new polyphenol 18, 1, 3-di-O-caffeoyl-5-O-(1-methoxyl-2-O-caffeoyl-4-maloyl)-quinic acid, together with 17 known compounds, was isolated and characterized. In conclusion, Compound 18 was a new caffeoyl maloyl quinic acid type polyphenol and showed desired vitro anti-oxidant activity. Compounds 1–5, 9, 10, 14, 15, and 17 were isolated from cultured cells of Saussurea involucrata for the first time.

关键词： cultured cells Saussurea involucrata Polyphenol Free radical scavenging activity

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Sequential extraction of RNA,DNA and protein from cultured cells of the same group

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World Journal of Methodology 2023年第5期13卷 484-491页

作者： Ying-Yu Cui Department of Cell Biology Institute of Medical GeneticsKey Laboratory of Arrhythmias of the Ministry of Education of ChinaTongji University School of MedicineShanghai 200331China

BACKGROUND Efficient extraction of nucleic acids and proteins(ENAP)from cells is a prerequisite for precise annotation of gene function,and has become laboratory routine for revealing the mysteries of ***,cell samples are often from different culture dishes,resulting in inevitable experimental errors and sometimes poor *** To explore a method to improve the efficiency of ENAP,minimizing errors in ENAP processes,enhancing the reliability and repeatability of subsequent experimental *** A protocol for the sequential isolation of RNA,DNA,and proteins from the same cultured HepG2 cells using RNAzol reagent is presented *** first step involves culturing HepG2 cells to the exponential phase,followed by the sequential isolation of RNA,DNA,and proteins from the same cultured cells in the second *** yield of nucleic acids and proteins is detected in the third step,and their purity and integrity are verified in the last *** The procedure takes as few as 3-4 d from the start to quality verification and is highly *** contrast to the existing kits and reagents,which are primarily based on independent isolation,this RNAzol reagent-based method is characterized by the sequential isolation of RNA,DNA,and proteins from the same cells,and therefore saves time,and has low cost and high *** The RNA,DNA,and proteins isolated using this method can be used for reverse transcription-polymerase chain reaction,polymerase chain reaction,and western blotting,respectively.

关键词： Sequential extraction Ribonucleic acid Deoxyribonucleic acid Protein cultured cells

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Effect of Transforming Growth Factor-β_2 on Phagocytosis in cultured Bovine Trabecular Meshwork cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2001年第4期21卷 318-320页

作者：曹阳魏厚仁笪邦红黄毅 Department of Ophthalmology Xiehe Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430022

The effect of transforming growth factor β 2 (TGF β 2) on phagocytosis in bovine trabecular meshwork cells in vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0.32 ng/ml, 1 ng/ml, 3.2 ng/ml TGF β 2 for 24 h, latex beads were added into the incubation medium, and the numbers of the latex beads in 20 adjacent cells were counted under a microscope 24 h later, after treatment with Wright's stain. Our results showed that the average numbers of the latex beads in the trabecular meshwork cells treated with TGF β 2 of different concentrations were 53.1±1.7 beads/cell, 56.4±2.9 beads/cell and 77.9±6.5 beads/cell respectinvely, in comparison with 45.5±3.3 beads/cell of the control group. TGF β 2 significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose dependent manner. TGF β 2 could promote the phagocytosis of bovine trabecular meshwork cells in vitro . It may be involved in the cellularity decrease of the trabecular meshwork in the patients of primary open angle glaucoma through promoting the phagocytosis of trabecular meshwork cells.

关键词： transforming growth factor trabecular meshwork cultured cells phagocytosis

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EFFECT OF QUERCETIN ON cultured HUMAN VASCULAR ENDOTHELIAL cells

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Academic Journal of Xi'an Jiaotong University 2000年第1期12卷 17-18,封四页

作者：林蓉刘俊田李旭陈葳 Center of Molecular Biology First Affiliated Hospital Xi'an Medical University Xi'an 710061 China

Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amount of ET-1 and PGI2 produced by VEC. VEC prollferation was assessed by tetrazolium(MTT) assay. Results Que increased the normal VEC prollferation at the concentration or 5, 2o, 4o, 8o, 1oompol/L and increased the production of PG12 and inhibits the release of ET by the normal VEC at the concentratiou or 5, 2o and 8ompol/L. Que at the concentration of 5, 2o and 8omol/L had no direct effect on morphology of the normal VEC. ConcIusion Que can stimulate the proliferation of VEC and inhibit tbe reIease of ET-1 and increase the formation of PGI2. The data suggest that Que might be beneficial for the prevention and treatment of vascular endothelial injury-related cardiovascular diseases, such as atherosclerosls and thromboembolism diseases.

关键词： quercetin vascular endothelium cultured cells

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Expression of Inositol 1,4,5-trisphosphate Receptor mRNA in Myocardium of Spontaneous Hypertension Rats and cultured Vascular Smooth Muscle cells of Rats

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Journal of Nanjing Medical University 2002年第2期16卷 75-79页

作者：刘乃丰张寄南耿茜杨笛董莉马文珠 Division of Cardiology Zhongda Hospital Southeast University Nanjing 210009 Research Institute of Cardiovascular Diseases NJMU Nanjing 210029 P. R. China

Objective\ To investigate expression of inositol 1,4,5 trisphosphate receptor (IP\-3R) mRNA on sacroplasmic reticular in myocardium of spontaneous hypertension rats (SHRs) and cultured vascular smooth muscle cells (VSMC) of rats and effects of perindopril and urapidil on them. Methods\ SHRs were orally given perindopril (1.0 mg·kg\+\{ 1\}·d\+\{ 1\}) or urapidil (15 mg·kg\+\{ 1\}·d\+\{ 1\}) for 24 weeks, respectively. Expression of IP\-3R mRNA was examined by semi quantitative reverse transcription polymers chain reaction (RT PCR) using three oligonuclotide primers for each subtype of IP\-3R with β actin as internal label. Results\ All subtypes of IP\-3R were expressed in myocardium of SHR, WKY and cultured VSMC. Expression of IP\-3R mRNA in left ventricle of SHR was markedly enhanced. Urapidil could down regulate expression of IP\-3R Ⅰand IP\-3R Ⅲ, perindopril slightly increased expression of IP\-3R Ⅱ and decreased expression of IP\-3R Ⅰand IP\-3R Ⅲ in myocardium of SHR. Conclusion\ Our results suggest that expression of IP\-3R mRNA in cardiovascular system could be regulated by urapidil and perindopril.

关键词： calcium release channel signal transduction inositol 1,4,5 trisphosphate receptor spontaneous hypertension rat vascular smooth muscle cultured cells polymers chain reaction

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Uptake of dopamine by rat hepatocytes in vitro

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Acta Pharmacologica Sinica 1994年第4期15卷 3-8页

作者： Simone WATTIAUX-DE CONINCK,Robert WATTIAUX Laboratoire de Chimie Physiologique Faculté de Médecine Facultés Universitaires Notre-Dame de la Paix B-5000 Namur Belgium Laboratoire de Chimie Physiologique Faculté de Médecine Facultés Universitaires Notre-Dame de la Paix B-5000 Namur Belgium

The present results showed that uptake of dopamine (DA) by rat isolated hep-atocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66. 8 μmol and Vmax of 52. 3 pmol·min-1/105 cells. The process was pH- and temperature-dependent and required an activation energy of 4. 12 kcal ·mol-1(Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal·mol -1(Q10 = 2. 0) in the range of 12.7 -39.0C. Cysteine residue having free thiol group was. unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport , but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transduci

关键词： liver cultured cells dopamine biological transport wasp venoms

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Quantitative proteomic determination of diethylstilbestrol action on prostate cancer

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Asian Journal of Andrology 2013年第3期15卷 413-420页

作者： Pierre Bigot Kevin Mouzat Muriel Bahut Nora Benhabiles Geraldine Cancel Tassin Abdel-Rahmene Azzouzi Olivier Cussenot Department of Urology Angers University Hospital Angers 49933 France CeRePP Tenon Hospital Paris 75970 France Department of Biochemistry N'imes University Hospital NTmes 30029 France Plate-Forme Technologique de Biotechnologie Moleculaire Angers University Angers 49933 France 5CEA DSV IRCM SREIT Laboratoire de Cancerologie Experimentale Fontenay-aux-Roses 92265 France

Diethylstilbestrol （DES） has a direct cellular mechanism inhibition on prostate cancer. Its action is independent from the oestrogen receptors and is preserved after a first-line hormonal therapy. We aimed to identify proteins involved in the direct cellular inhibition effects of DES on prostate cancer. We used a clonogenic assay to establish the median lethal concentration of DES on 22RV1 cells. 22RV1 cells were exposed to standard and DES-enriched medium. After extraction, protein expression levels were obtained by two-dimensional differential in-gel electrophoresis （2D-DIGE） and isotope labelling tags for relative and absolute quantification （iTRAQ）. Proteins of interest were analysed by quantitative RT-PCR and western blotting. The differentially regulated proteins （P〈0.01） were interrogated against a global molecular network based on the ingenuity knowledge base. The 2D-DIGE analyses revealed DES-induced expression changes for 14 proteins （〉 1.3 fold; P〈0.05）. The iTRAQ analyses allowed the identification of 895 proteins. Among these proteins, 65 had a modified expression due to DES exposure （i.e., 23 overexpressed and 42 underexpressed）. Most of these proteins were implicated in apoptosis and redox processes and had a predicted mitochondrial expression. Additionally, ingenuity pathway analysis placed the OAT and HSBP1 genes at the centre of a highly significant network. RT-PCR confirmed the overexpression of OAT （P=0.006） and HSPB1 （P=0.046）.

关键词： cultured cells DES diethylstilbestrol ingenuity pathway analysis isotope labelling mass spectrometry prostate cancer proteomics

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Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第1期23卷 85-87页

作者：曹阳魏厚仁笪邦红李忠玉 DepartmentofOphthalmology XieheHospitalTongjiMedicalCollegeHuazhongUniversityofScienceandTechnology DepartmentofEmergency XieheHospitalTongjiMedicalCollegeHuazhongUniversityofScienceandTechnology DepartmentofAnatomy TongjiMedicalCollegeHuazhongUniversityofScienceandTechnology

Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.

关键词： human trabecular meshwork cultured cells transforming growth factor reverse transcriptase-polymerase chain reaction immunohistochemistry

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Analysis of the Interface between Primary and Secondary Metabolism in Catharanthus roseus Cell Cultures Using ^13C-Stable Isotope Feeding and Coupled Mass Spectrometry

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Molecular Plant 2013年第2期6卷 581-584页

作者： Carla Antonio Natali R. Mustafa Sonia Osorio Takayuki Tohge Patrick Giavalbco Lothar Willmitzer Heiko Rischer Kirsi-Marj Oksman-Caldentey Robert Verpoorte Alisdair R. Fernie Central Metabolism Group Max Planck Institute of Molecular Plant Physiology Am Muehlenberg 1 D-14476 Potsdam-Golm Germany Natural Products Laboratory Institute of Biology Leiden University Einsteinweg 55 2300 RA Leiden The Netherlands Present address: Departamento de Biologia Molecular y Bioquimica Universidad de M.~laga 29071 M=~laga Spain Genes and Small Molecules Group Max Planck Institute of MolecularPlant Physiology Am Muehlenberg 1 D-14476 Potsdam-Golm Germany VTT Technical Research Centre of Finland PO Box 1000 F1-02044 VTT (Espoo) Finland

Dear Editor, The tropical plant Madagascar periwinkle Catharanthus roseus （L.） *** is a rich source of plant-derived medicinal ter-penoid indole alkaloids （TIAs）, including the anti-hypertensive ajmalicine, the sedative compound serpentine, and the anti-cancer drugs vinblastine and vincristine. However, the latter two compounds are produced in C. roseus plants only in very low amounts. Elicitors such as hormones （e.g. jasmonates or salicylic acid） activate plant natural defense responses, includ-ing increased secondary metabolite production （EI-Sayed and Verpoorte, 2007; Lackman et al.. 2011）.

关键词： Secondary Metabolism Terpenes jasmonate biosynthesis Pathway Salicylic Acid cultured cells Interfaces Cisplatin/Etoposide/Methotrexate Catharanthus roseus Interface

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