scholarly journals 181 AN ULTRACELLULAR LOOK AT LIGHT-TREATED IPT-TRANSGENIC TOBACCO

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 455a-455
Author(s):  
Sandra L. Barbour ◽  
Dennis R. Decoteau
Keyword(s):  
Transgenic Tobacco ◽  
Chloroplast Development ◽  
Polyclonal Antibodies ◽  
Red Light ◽  
Rabbit Antiserum ◽  
Isopentenyl Transferase ◽  
Transmission Electron ◽  
Sds Page ◽  
Transgenic Cells ◽  
Cytokinin Synthesis

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development. amaranthin synthesis, seed germination, photomorphogenesis). It is unclear. however, if and how these two systems interact. To determine the effects of phytochrome activity on cytokinin synthesis and ultracellular plant development, we utilized tobacco transformed with the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene. This gene encodes for isopentenyl transferase (iptase) which is an enzyme active in cytokinin biosysthesis. Ipt-transgenic tobacco cultures were treated with end-of-day red or far-red light for 15 minutes. After 15-30 days of treatment, the plant tissue was harvested and ipt expression was verified by SDS-PAGE and western blot analysis. Polyclonal antibodies specific to iptase were used as a primary antibody. Colloidal gold conjugated to goat. anti-rabbit antiserum served as an electron dense, secondary antibody and a probe to light-influenced iptase synthesis and distribution within the cell. A Hitachi 600AB transmission electron microscope was used to determine the influence of phytochrome/light treatments on the ultrastructure of ipr-transgenic cells.

scholarly journals IPTASE SYNTHESIS AND LIGHT MEDIATION IN TRANSGENIC TOBACCO

HortScience ◽  
1994 ◽  
Vol 29 (7) ◽  
pp. 737e-737
Author(s):  
Sandra L. Barbour ◽  
Margaret J. McMahon ◽  
John J. Frett ◽  
Dennis R. Decoteau
Keyword(s):  
Plant Growth ◽  
Transgenic Tobacco ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Red Light ◽  
Microscopic Analysis ◽  
Cellular Level ◽  
Cytokinin Activity ◽  
Electron Microscopic ◽  
Cytokinin Biosynthesis

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis. seed germination, photomorphogenesis). It is unclear, however, if and how these two systems interact. As a beginning step to determine cytokinin-phytochrome interactions, we developed a strategy utilizing ipt -transgenic tobacco in phytochrome/light treatment investigations. The sour-cc of the ipt gene was Agrobacterium tumefaciens Ti plasmid 15955. This gene encodes for isopentenyl transferase which is an enzyme active in cytokinin biosynthesis. Ipt -transgenic tobacco cultures (grown on MS medium supplemented with kanamycin but no plant growth regulators) were treated with end-of-day red or far-red light for 15 minutes. After 30 days of treatment, the plant tissue was harvested and either homogenized for SDS-PAGE or fixed for transmission electron microscopic analysis. Results from immuno-gold labelling using polyclonal antibodies specific to iptase will he used to Indicate the influence of phytochrome on cytokinin activity. Also, structural changes at the ultra-cellular level will be determined.

scholarly journals PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST IPTASE

HortScience ◽  
1993 ◽  
Vol 28 (4) ◽  
pp. 257F-257
Author(s):  
Sandra L. Barbour ◽  
John J. Frett
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusion Protein ◽  
New England ◽  
Polyclonal Antibodies ◽  
Isopentenyl Transferase ◽  
Cytokinin Biosynthesis ◽  
Western Blots ◽  
Sds Page ◽  
England Biolabs ◽  
Secondary Antibodies

Isopentenyl transferase, encoded by the ipt gene of Agrobacterium tumefaciens T-DNA, is an enzyme active in cytokinin biosynthesis. The ipt gene was cloned into the pMAL-c2 vector (New England Biolabs, Beverly, MA) and expressed as a fusion protein. The production of this fusion protein was induced by a 2 hour exposure to IPTG. The fusion protein was then purified by a mini-aggregate procedure and visualized by SDS-PAGE. To verify that the correct protein was purified, antibodies specific to the conserved region of the fusion protein were used to probe a western blot. Secondary antibodies were biotinylated. Rabbits were immunized to raise polyclonal antibodies against iptase. Using a slot format blotting apparatus, serum was titered. These antibodies will be used to probe western blots from transgenic plants transformed with various ipt constructs.

Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

2005 ◽  
Vol 288 (1) ◽  
pp. C141-C147 ◽  
Author(s):  
Yu-Hua Zhang ◽  
Diego Alvarez de la Rosa ◽  
Cecilia M. Canessa ◽  
John P. Hayslett
Keyword(s):  
Sodium Channel ◽  
Sodium Transport ◽  
Polyclonal Antibodies ◽  
Hormonal Stimulation ◽  
Α Subunit ◽  
A6 Cells ◽  
Kinase C ◽  
Sds Page ◽  
Protein Kinase C Inhibitor ◽  
Synergistic Increase

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with 35S or 32P. When immune complexes were resolved on SDS-PAGE, the α-subunit migrated at 85 and 65 kDa, the β-subunit at 115 and 100 kDa, and the γ-subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The α-subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of α is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of α-, β-, and γ-subunits approximately twofold. Exposure to 1 μM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport.

Notizen: Changes in the Iron and Phosphorus Content of Stroma Inclusions during Etioplast-Chloroplast Development in Nicotiana

1977 ◽  
Vol 32 (1-2) ◽  
pp. 139-142
Author(s):  
B. Jülich ◽  
G. Gliem ◽  
A. G. S Jánossy
Keyword(s):  
Conformational Changes ◽  
Phosphorus Content ◽  
Chloroplast Development ◽  
Prolamellar Body ◽  
High Electron ◽  
Formation Processes ◽  
Thin Sections ◽  
X Ray ◽  
Transmission Electron ◽  
Phosphorus Containing

Conformational changes of the thylakoid arrangement during light-dependent etioplast-chloroplast development in cotyledons of Nicotiana clevelandii X N. glutinosa are correlated with a decrease of the iron and phosphorus content in electron-dense stroma inclusions. Parallel to the transformation of the prolamellar body and the stacking process of the thylakoids, both the iron and phosphorus content of the inclusions were found to be reduced. Their elemental composition was analysed by means of the energy-dispersive X-ray microanalysis. Due to their high electron-density these stroma inclusions can be observed by conventional transmission electron microscopy in unstained thin-sections from exclusively glutaraldehyde-fixed material. They seem to be involved in membrane formation processes concomitant with the dispersal of the prolamellar bodies. Thus, the iron and phosphorus containing inclusions were found either closely surrounded by membranes or in the intralamellar space of plastids from plantlets illuminated for 1 - 8 hours. In chloroplasts (illumination period 12 -24 hours) no connections between these inclusions and the thylakoids were noticed.

PLATELETS OF A PATIENT LACKING GLYCOPROTEINS lib AND Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR COLLAGEN

1987 ◽  
Author(s):  
J L McGregor ◽  
L McGregor ◽  
M Hans ◽  
A Sayegh ◽  
M C Trzeeiak ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Two Dimensional ◽  
Binding Studies ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Sds Page ◽  
Bleeding Episodes ◽  
High Concentrations ◽  
Induced Aggregation

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.

VEGF-induced mobilization of caveolae and increase in permeability of endothelial cells

2002 ◽  
Vol 282 (5) ◽  
pp. C1053-C1063 ◽  
Author(s):  
Jun Chen ◽  
Filip Braet ◽  
Sergey Brodsky ◽  
Talia Weinstein ◽  
Victor Romanov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Electrical Resistance ◽  
Polyclonal Antibodies ◽  
Endothelial Permeability ◽  
Green Fluorescence Protein ◽  
Vascular Endothelial ◽  
Transmission Electron ◽  
Glomerular Epithelial Cells ◽  
Fluorescence Protein ◽  
Endothelial Growth Factor

Glomerular epithelial cells (GEC) are a known site of vascular endothelial growth factor (VEGF) production. We established immortalized rat GEC, which retained the ability to produce VEGF. The isoforms expressed by GEC were defined as VEGF-205, -188, -120, and -164. The electrical resistance of endothelial cells cultured on GEC-conditioned matrix, an indicator of the permeability of monolayers to solutes, was significantly increased by the treatment with the neutralizing polyclonal antibodies to VEGF and decreased by VEGF-165. Transfection of endothelial cells with green fluorescence protein-caveolin construct and intravital confocal microscopy showed that VEGF results in a rapid appearance of transcellular elongated structures decorated with caveolin. Transmission electron microscopy of endothelial cells showed that caveolae undergo rapid internalization and fusion 30 min after application of VEGF-165. Later (36 h), endothelial cells pretreated with VEGF developed fenestrae and showed a decrease in electrical resistance. Immunoelectron microscopy of glomeruli confirmed VEGF localization to podocytes and in the basement membrane. In summary, immortalized GEC retain the ability to synthesize VEGF. Matrix-deposited and soluble VEGF leads to the enhancement of caveolae expression, their fission and fusion, formation of elongated caveolin-decorated structures, and eventual formation of fenestrae, both responsible for the increase in endothelial permeability.

scholarly journals Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation

1999 ◽  
Vol 337 (3) ◽  
pp. 531-536 ◽  
Author(s):  
Eduardo LÓPEZ-HUERTAS ◽  
Francisco J. CORPAS ◽  
Luisa M. SANDALIO ◽  
Luis A. DEL RÍO
Keyword(s):  
Cytochrome C ◽  
Electron Donor ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Monodehydroascorbate Reductase ◽  
Peroxisomal Membrane ◽  
Cytochrome C Reductase ◽  
Ferricyanide Reductase ◽  
Sds Page ◽  
O2 Production

The production of superoxide radicals (O2-•) and the activities of ferricyanide reductase and cytochrome c reductase were investigated in peroxisomal membranes from pea (Pisum sativum L.) leaves using NADH and NADPH as electron donors. The generation of O2-• by peroxisomal membranes was also assayed in native polyacrylamide gels using an in situ staining method with NitroBlue Tetrazolium (NBT). When peroxisomal membranes were assayed under native conditions using NADH or NADPH as inducer, two different O2-•-dependent Formazan Blue bands were detected. Analysis by SDS/PAGE of these bands demonstrated that the NADH-induced NBT reduction band contained several polypeptides (PMP32, PMP61, PMP56 and PMP18, where PMP is peroxisomal membrane polypeptide and the number indicates molecular mass in kDa), while the NADPH-induced band was due exclusively to PMP29. PMP32 and PMP29 were purified by preparative SDS/PAGE and electroelution. Reconstituted PMP29 showed cytochrome c reductase activity and O2-• production, and used NADPH specifically as electron donor. PMP32, however, had ferricyanide reductase and cytochrome c reductase activities, and was also able to generate O2-• with NADH as electron donor, whereas NADPH was not effective as an inducer. The reductase activities of, and O2-• production by, PMP32 were inhibited by quinacrine. Polyclonal antibodies against cucumber monodehydroascorbate reductase (MDHAR) recognized PMP32, and this polypeptide is likely to correspond to the MDHAR reported previously in pea leaf peroxisomal membranes.

Luminescent properties of Y2O3:Eu3+ nanophosphor prepared from urea added precursor using flame spray pyrolysis

2009 ◽  
Vol 24 (8) ◽  
pp. 2584-2588 ◽  
Author(s):  
Jae Seok Lee ◽  
Se Jin Kim ◽  
Tae Kon Kim ◽  
Rajiv K. Singh ◽  
Madhav B. Ranade
Keyword(s):  
Electron Microscopy ◽  
Spray Pyrolysis ◽  
Red Light ◽  
Luminescent Properties ◽  
Flame Spray Pyrolysis ◽  
Cubic Phase ◽  
Flame Spray ◽  
X Ray Diffraction ◽  
Synthesis Conditions ◽  
Transmission Electron

Y2O3:Eu3+ nanophosphor was synthesized by flame spray pyrolysis (FSP) from urea added nitrate based liquid precursor. In this study, urea serves as fuel and subsequently provides additional heat in the flame zone during the synthesis of phosphor particles. The end product shows cubic phase Y2O3:Eu3+ nanophosphor successfully prepared by FSP without heat treatment. The influence of synthesis conditions such as different mol of urea and nitrate source materials in aqueous solution, and doping concentration on luminescent properties, were investigated. The characteristics of nanophosphor such as crystallinity and morphology under various experiments of conditions were carried out by x-ray diffraction (XRD) and field emission-scanning electron microscopy (FE-SEM). The particle size of product was found to be in the range of 20–30 nm from transmission electron microscopy (TEM). In photoluminescence (PL) properties, Y2O3:Eu3+ nanophosphor emitted red light with a peak wavelength of 609 nm when excited with 398 nm wavelength photons.

scholarly journals Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy.

1986 ◽  
Vol 103 (2) ◽  
pp. 393-404 ◽  
Author(s):  
R R Bruns ◽  
W Press ◽  
E Engvall ◽  
R Timpl ◽  
J Gross
Keyword(s):  
Immunoelectron Microscopy ◽  
Polyclonal Antibodies ◽  
Structural Characteristics ◽  
Rabbit Antiserum ◽  
Type Vi Collagen ◽  
Thin Sections ◽  
Prolonged Incubation ◽  
Fibroblast Cultures ◽  
Band Region ◽  
Wide Range

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.

Immunolocalization of a proteinase of the sap-staining fungus Ophiostoma piceae using antibodies to proteinase K

1995 ◽  
Vol 73 (10) ◽  
pp. 1604-1610 ◽  
Author(s):  
C. Hoffert ◽  
S. Gharibian ◽  
C. Breuil ◽  
D. L. Brown
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Cell Walls ◽  
Polyclonal Antibodies ◽  
Immunogold Labelling ◽  
Proteinase K ◽  
Igg Antibodies ◽  
Extracellular Proteinase ◽  
Ophiostoma Piceae ◽  
Transmission Electron

Polyclonal antibodies were raised against proteinase K and were used to immunolocalize the major extracellular proteinase of the sap-staining fungus Ophiostoma piceae (Münch) H. and P. Sydow. Immunodot blotting showed that the IgG antibodies recognized both enzymes but reacted more strongly with proteinase K than with the O. piceae proteinase. Immunogold labelling and transmission electron microscopy revealed that the O. piceae proteinase was localized in the cell walls of O. piceae grown either in liquid media or wood. Key words: Ophiostoma piceae, proteinase, immunogold labelling, transmission electron microscopy, antibody, proteinase K.

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