FEBS Journal

  1742-464X

  1432-1033

  Anh Quốc

Cơ quản chủ quản:  Wiley-Blackwell Publishing Ltd , WILEY

Lĩnh vực:
Cell BiologyMolecular BiologyBiochemistry

Các bài báo tiêu biểu

Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase
Tập 47 Số 3 - Trang 469-474 - 1974
Stefan L. Marklund, Gudrun MARKLUND
The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6.The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2· ‐independent mechanisms rapidly become dominant.Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism.A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.A plausible explanation is given for the non‐competitive part of the inhibition of catechol O‐methyltransferase brought about by pyrogallol.
A Film Detection Method for Tritium‐Labelled Proteins and Nucleic Acids in Polyacrylamide Gels
Tập 46 Số 1 - Trang 83-88 - 1974
William M. Bonner, Ronald A. Laskey
A simple method is described for detecting 3H in polyacrylamide gels by scintillation autography (fluorography) using X‐ray film. The gel is dehydrated in dimethyl sulphoxide, soaked in a solution of 2,5‐diphenyloxazole (PPO) in dimethylsulphoxide, dried and exposed to RP Royal “X‐Omat” film at ‐70 °C. Optimal conditions for each step are described. β‐particles from 3H interact with the 2,5‐diphenyloxazole emitting light which causes local blackening of an X‐ray film. The image produced resembles that obtained by conventional autoradiography of isotopes with higher emission energies such as 14C. 3000 dis. 3H/min in a band in a gel can be detected in a 24‐h exposure. Similarly 500 dis./min can be detected in one week.When applied to the detection of 35S and 14C in polyacrylamide gels, this method is ten times more sensitive than conventional autoradiography. 130 dis. 35S or 14C/min in a band in a gel can be detected in 24 h.
Quantitative Film Detection of 3H and 14C in Polyacrylamide Gels by Fluorography
Tập 56 Số 2 - Trang 335-341 - 1975
Ronald A. Laskey, Anthony D. Mills
An Efficient mRNA‐Dependent Translation System from Reticulocyte Lysates
Tập 67 Số 1 - Trang 247-256 - 1976
Hugh R.B. Pelham, Richard J. Jackson
A simple method is described for converting a standard rabbit reticulocyte cell‐free extract (lysate) into an mRNA‐dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol‐bis(2‐aminoethylether)‐N,N′‐tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full‐sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.
Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity
Tập 267 Số 17 - Trang 5421-5426 - 2000
John O’Brien, Ian D. Wilson, Terry C. Orton, François Pognan
We show here the identity of Alamar Blue as resazurin. The ‘resazurin reduction test’ has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.
A Protein Sequenator
Tập 1 Số 1 - Trang 80-91 - 1967
Pehr Edman, Geoffrey S. Begg
The protein sequenator is an instrument for the automatic determination of amino acid sequences in proteins and peptides. It operates on the principle of the phenylisothiocyanate degradation scheme. The automated process embraces the formation of the phenylthiocarbamyl derivative of the protein and the splitting off of the N‐terminal amino acid as thiazolinone. The degradation proceeds at a rate of 15.4 cycles in 24 hours and with a yield in the individual cycle in excess of 98%. The material requirements are approximately 0.25 μmoles of protein. The thiazolinones are converted to the corresponding phenylthiohydantoins in a separate operation, and the latter identified by thin layer chromatography. The process has been applied to the whole molecule of apomyoglobin from the humpback whale, and it has been possible to establish the sequence of the first 60 amino acids from the N‐terminal end.
Purification of Mouse Immunoglobulin Heavy‐Chain Messenger RNAs from Total Myeloma Tumor RNA
Tập 107 Số 2 - Trang 303-314 - 1980
Charles Auffray, François Rougeon
A procedure is described for the large‐scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H‐chain mRNAs were purified by oligo(dT)‐cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high‐resolution preparative gel electrophoresis under non‐denaturing conditions. γ2a and α H‐chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L‐chain mRNAs sedimented as 12‐S species. H‐chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L‐chain mRNA and residual 18‐S rRNA, and migrated as single components of 1900 ± 50 nucleotides on analytical denaturing gels. The partially purified H‐chain mRNAs were translated into major components of molecular weights of 56000 (γ2a) and 60000 (α) in an mRNA‐dependent rabbit reticulocyte lysate, whereas L‐chain mRNAs yielded polypeptides of molecular weights of 25000 (λ) and 27000 (к). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H‐chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (γ2a), 62% (α), for H‐chain mRNAs and 60% (к), for L‐chain mRNAs.
Physiological functions of thioredoxin and thioredoxin reductase
Tập 267 Số 20 - Trang 6102-6109 - 2000
Elias S.J. Arnér, Arne Holmgren
Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione‐disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin–thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF‐κB or the Ref‐1‐dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co‐cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70‐kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112–130 kDa), selenium‐dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C‐terminal elongation with a cysteine–selenocysteine sequence forming a redox‐active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.
Patterns of Amino Acids near Signal‐Sequence Cleavage Sites
Tập 133 Số 1 - Trang 17-21 - 1983
Gunnar von Heijne
According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side‐chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made.
Strategies of antioxidant defense
Tập 215 Số 2 - Trang 213-219 - 1993
Helmut Sies
Cellular protection against the deleterious effects of reactive oxidants generated in aerobic metabolism, called oxidative stress, is organized at multiple levels. Defense strategies include three levels of protection; prevention, interception, and repair. Regulation of the antioxidant capacity includes the maintenance of adequate levels of antioxidant and the localization of antioxidant compounds and enzymes. Short‐term and long‐term adaptation and cell specialisation in these functions are new areas of interest. Control over the activity of prooxidant enzymes, such as NADPH oxidase and NO synthases, is crucial. Synthetic antioxidants mimic biological strategies.