The small domain of cytochromeffrom the psychrophileChlamydomonas raudensisUWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochromefin the mesophileChlamydomonas reinhardtii

2007 ◽  
Vol 85 (5) ◽  
pp. 616-627 ◽  
Author(s):  
Loreta Gudynaite-Savitch ◽  
Christelle Loiselay ◽  
Leonid V. Savitch ◽  
John Simmonds ◽  
Susanne E. Kohalmi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Electron Transport ◽  
Molecular Mass ◽  
Chloroplast Transformation ◽  
Apparent Molecular Mass ◽  
Cytochrome F ◽  
Structure Stability ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Small Domain

Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e–/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.

scholarly journals Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils

1988 ◽  
Vol 252 (1) ◽  
pp. 143-149 ◽  
Author(s):  
A K Campbell ◽  
A K Patel ◽  
Z S Razavi ◽  
F McCapra
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Cell Activation ◽  
Membrane Attack Complex ◽  
Living Cells ◽  
Human Neutrophils ◽  
Protein Synthesis Inhibitor ◽  
Chemotactic Peptide ◽  
Prosthetic Group ◽  
Synthesis Inhibitor

1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.

The reduction of trimethylarsine oxide by Candida humicola

1981 ◽  
Vol 27 (8) ◽  
pp. 773-778 ◽  
Author(s):  
A. W. Pickett ◽  
Barry C. McBride ◽  
W. R. Cullen ◽  
H. Manji
Keyword(s):  
Protein Synthesis ◽  
Electron Transport ◽  
Cell Concentration ◽  
Protein Synthesis Inhibitor ◽  
Optimum Temperature ◽  
Synthesis Inhibitor ◽  
Optimum Ph ◽  
Transport Inhibitors ◽  
Biological Transformation ◽  
Trimethylarsine Oxide

Trimethylarsine oxide, a probable intermediate in the biological transformation of arsenate, was reduced to volatile trimethylarsine by Candida humicola. A simple assay for the rate of trimethylarsine production from trimethylarsine oxide by the fungus was developed. The optimum pH for the reduction was determined as 5.1–5.2, and the optimum temperature was 40 °C. The rate of reduction was directly proportional to cell concentration and followed Michaelis–Menten type kinetics. There was almost no trimethylarsine produced by heated or broken cells. The reaction was inhibited by a number of electron transport inhibitors and uncouplers of oxidative phosphorylation including cyanide, azide, and 2,4-dinitrophenol. The rate of reduction was modified by arsenate, methylarsonate, dimethylarsinate, selenate, and tellurate. Preincubation of cells with trimethylarsine oxide increased the rate of reduction 69-fold; this increase in activity was blocked if the cells were incubated with the protein synthesis inhibitor cycloheximide.

scholarly journals Fibrinogenolysis by a neutrophil membrane protease generates an A α 1–21 fragment

1994 ◽  
Vol 298 (3) ◽  
pp. 689-695 ◽  
Author(s):  
S L Kelly ◽  
S A Adams ◽  
S C Robson ◽  
R E Kirsch ◽  
E G Shephard
Keyword(s):  
Amino Acids ◽  
Molecular Mass ◽  
Anticoagulant Activity ◽  
Acute Phase Reactant ◽  
Apparent Molecular Mass ◽  
Gamma Chain ◽  
Reactive Protein ◽  
C Terminus ◽  
N Terminus

The 600 kDa neutrophil membrane neutral protease, which had been shown to generate bioactive peptides from the acute-phase reactant C-reactive protein, has now been shown to have fibrinogenolytic activity that is distinct from fibrinogenolysis by plasmin and neutrophil lysosomal enzymes. This protease gradually reduces the apparent molecular mass of fibrinogen (340 kDa) to non-clottable products and generates terminal products with apparent molecular mass values of 270 kDa, 200 kDa, 100 kDa and less than 40 kDa through cleavage of all three of the constituent chains. Characteristics of fibrinogenolysis by this neutrophil protease are cleavage of the bond between amino acids valine and glutamic acid at positions 21 and 22 respectively from the N-terminus of the A alpha chain to release an A alpha 1-21 peptide, digestion of the B beta chain at positions within the C-terminus, and proteolysis of the bond between amino acids isoleucine and glycine at positions 394 and 395 respectively from the N-terminus of the gamma chain. This generates products that lack anticoagulant activity. The thrombin clotting time of the product with an apparent molecular mass of 330 kDa was prolonged, although clot formation was still observed. Loss of coagulability and inability to clot was found with further degradation of fibrinogen to an apparent molecular mass of 290 kDa. Activity of this neutrophil membrane protease in vivo could be important for the regulation of fibrin deposition at sites of inflammation, and may contribute to the reported plasma levels of the A alpha 1-21 peptide.

Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids

1986 ◽  
Vol 55 (03) ◽  
pp. 369-374 ◽  
Author(s):  
Raffaele De Caterina ◽  
Babette B Weksler
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Arachidonic Acid Metabolism ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Prolonged Incubation ◽  
Dependent Inhibition ◽  
Human Thrombin ◽  
Umbilical Vein Endothelial Cells

SummaryTo learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.

PROTECTIVE EFFECT OF POLYPEPTIDE AND AMINO ACIDS ON THE DEVELOPMENT OF THE NERVOUS TISSUE CULTURE IN THE PRESENCE OF CYCLOPHOSPHAMIDE

2017 ◽  
pp. 22-26
Author(s):  
V. B. Dolgo-Saburov ◽  
N. I. Chalisova ◽  
L. V. Lyanginen ◽  
E. S. Zalomaeva
Keyword(s):  
Amino Acids ◽  
Cell Proliferation ◽  
Tissue Culture ◽  
Protective Effect ◽  
Organotypic Culture ◽  
Inhibitory Effect ◽  
Mustard Gas ◽  
Synthesis Inhibitor ◽  
Combined Administration ◽  
The Central Nervous System

In an organotypic culture, an investigation was conducted into combined effects of cyclophosphamide DNA as synthesis inhibitor used to model a resorptive action of mustard gas, and cortexin polypeptide or each of 20 encoded amino acids on the development of cell proliferation in cerebral cortex explants of the rat. The combined administration of cyclophosphamide together with cortexin or with each of the 20 encoded amino acids, except glycine, showed suppression of the cytostatic agent inhibitory effect. Thus, cortexin and amino acids have a protective effect on cell proliferation in the tissue culture of the central nervous system under the action of mustardlike substances.

scholarly journals Effect of emetine, a protein synthesis inhibitor, on larval tail resorption in the ascidian Halocynthia roretzi

2006 ◽  
Vol 23 (2) ◽  
pp. 43-46
Author(s):  
Kiyotaka Matsumura ◽  
Manami Nagano ◽  
Sachiko Tsukamoto ◽  
Haruko Kato ◽  
Nobuhiro Fusetani
Keyword(s):  
Protein Synthesis ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
Halocynthia Roretzi

scholarly journals The Presence and Characterization of a virF Gene on Agrobacterium vitis Ti Plasmids

1998 ◽  
Vol 11 (5) ◽  
pp. 429-433 ◽  
Author(s):  
B. Schrammeijer ◽  
J. Hemelaar ◽  
P. J. J. Hooykaas
Keyword(s):  
Amino Acids ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Tumor Formation ◽  
Nicotiana Glauca ◽  
Agrobacterium Vitis ◽  
Ti Plasmids

Octopine and nopaline strains of Agrobacterium tumefaciens differ in their ability to induce tumors on Nicotiana glauca. The presence of a virF locus on the octopine Ti plasmid makes N. glauca a host plant for these strains, indicating that the VirF protein is a host-range determinant. Here we show the presence of a virF locus not only on the Agrobacterium vitis octopine/cucumopine plasmids pTiAg57 and pTiTm4, but also on the nopaline Ti plas-mids pTiAT1, pTiAT66a, and pTiAT66b. On the octopine Ti plasmids from A. tumefaciens the virF gene is located between the virE locus and the left border of the T-region. In contrast, the virF gene on Ti plasmids of A. vitis is located at the very left end of the vir-region near the virA locus. The virF gene of pTiAg57 has been sequenced and codes for a protein of 202 amino acids with a molecular mass of 22,280 Da. Comparison showed that the virF gene from A. vitis strain Ag57 is almost identical to that from A. tumefaciens octopine strains. The transcription of the pTiAg57 virF is inducible by the plant phenolic compound acetosyringone through the presence of a vir-box consensus sequence in its promoter region. The VirF protein from pTiAg57 can complement octopine A. tumefaciens strains deleted for virF as shown by tumor formation on N. glauca.

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