Biofactors - Volume 6, issue 3

Authors: Liu, Song‐Yuan | Stadtman, Thressa C.

Article Type: Research Article

Abstract: Selenophosphate synthetase catalyzes the formation of monoselenophosphate (SePO_{3}^{3-} ) from ATP and selenide (reaction 1). \begin{equation} {\rm ATP + HSe^{-}} \rightarrow {\rm SePO_{3}^{3-} + PO_{4}^{3-} + AMP} \end{equation} In one assay frequently used, [8‐^{14} C]AMP formation from [8‐^{14} C]ATP is estimated after separation of the nucleotides by thin‐layer chromatography. An alternative non‐radioactive assay in which the AMP product is estimated using AMP deaminase is described. The highly oxygen‐labile selenophosphate product can be estimated in an assay employing [\gamma ‐^{32} P]ATP. The ^{32} P‐labeled selenophosphate is converted to [^{32} P]orthophosphate …by treatment with iodine and estimated after removal of residual [^{32} P]ATP on charcoal. Show more

Citation: Biofactors, vol. 6, no. 3, pp. 305-309, 1997

Authors: Steinert, Peter | Ahrens, Marion | Gross, Gerhard | Flohé, Leopold

Article Type: Research Article

Abstract: An 11‐day embryonic Swiss Webster/NIH mouse cDNA library was screened with a partial murine selenoprotein P cDNA probe and a murine selenoprotein‐P‐type cDNA The accession number of the nucleic acid sequence data for murine selenoprotein P in the EMBL Data Library is X99807. clone of 2075 bp lenght was obtained. The clone contained a 5' ‐leader sequence of 132 bp length, the selenoprotein P coding frame, and 803 base pairs in the 3' untranslated region. Alignment and RNA folding studies revealed the presence of two well conserved selenocysteine inserting motifs in the 3' …flanking region. The deduced polypeptide sequence comprises 380 residues including ten selenocysteines. Identical amino acid residues in homologous positions are 86%, 71%, and 64% when compared to the previously reported selenoprotein P sequences of rat, man, and cattle, respectively. The comparatively low similarity between the selenoprotein P sequences reported so far leaves open the question whether they belong to the same molecular clade. The accession number of the nucleic acid sequence data for murine selenoprotein P in the EMBL Data Library is X99807. Show more

Keywords: Mouse, selenoprotein P, cDNA, sequencing

Citation: Biofactors, vol. 6, no. 3, pp. 311-319, 1997

Authors: Han, Derick | Handelman, Garry | Marcocci, Lucia | Sen, Chandan K. | Roy, Sashwati | Kobuchi, Hirotsugu | Tritschler, Hans J. | Flohé, Leopold | Packer, Lester

Article Type: Research Article

Abstract: Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells, human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by …the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the x_{\rm c}^{-} transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, \gamma ‐glutamylcysteine synthetase, which is regulated by uptake‐limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells. Show more

Keywords: ASC transporter, cysteine uptake, Jurkat cells, peripheral blood lymphocytes, thiols, x_{\rm c}^{-} transporter

Citation: Biofactors, vol. 6, no. 3, pp. 321-338, 1997

Authors: Nguyen‐Le, X.K. | Brière, N. | Corcos, J.

Article Type: Research Article

Abstract: A model previously developed in our laboratory to culture rat prostate explants in serum‐free chemically‐defined medium was used to evaluate the direct influence of potential regulators. The aim of the present work was to verify the effects of insulin (I) and transferrin (Tr), two hormones considered as essential in other serum‐free culture systems, and three androgenic hormones, since the prostate is known to be androgen‐dependent. Explants of rat prostate were cultured for five days in serum‐free Leibovitz’s L‐15 medium (37^\circ C, 95% air – 5% CO_{2} ). The addition of Tr (50 \mu g/ml) had no effect, …but I (5 \mu g/ml) significantly increased DNA synthesis. This influence was amplified by combination of the two hormones. However, protein synthesis was only slightly stimulated. Testosterone (T) or androstanediol significantly increased DNA synthesis when compared to corresponding control values at five days. In combination with I plus Tr, each hormone showed potentiated effects, particularly T with a twofold increase over day 0 values. When dihydrotestosterone was added singly, the incorporation of ^{3} H‐thymidine was stimulated by 300% over control values at five days, and by 100% over values in uncultured explants. This influence was maximal since it was not improved by I plus Tr. Protein synthesis was increased significantly by the triple combination. In addition, each androgen as well as the combination of I plus Tr had a positive influence on explant morphology. The above conditions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors in prostate metabolism and to eventually identify putative cancer markers. Show more

Citation: Biofactors, vol. 6, no. 3, pp. 339-349, 1997

Authors: Marcocci, Lucia | Flohé, Leopold | Packer, Lester

Article Type: Research Article

Abstract: Human thioredoxin reductase was recently shown to contain a TGA encoded selenocysteine residue at the penultimate position of its amino acid chain. Depending on the availability of selenium during biosynthesis, an authentic selenocysteine‐containing or a selenium‐free enzyme truncated at the penultimate position is expected to be formed. Correspondingly, the enzymatic activity should be altered by selenium restriction, if the selenocysteine residue is functionally important. In order to check the catalytic role of the selenocysteine residue, four different human cell lines were grown in selenium deficient media or with adequate selenium supplementation (40 nM sodium selenite) and thioredoxin reductase activity was …measured as NADPH‐dependent DTNB reduction or thioredoxin‐mediated insulin reduction. Thioredoxin reductase activities, like glutathione peroxidase activities, were consistently higher in selenium supplemented cells, whereas glutathione reductase activity was not affected by the selenium. The dose‐response was similar for thioredoxin reductase and glutathione peroxidase, but the recovery of glutathione peroxidase activity upon selenium supplementation was faster than with thioredoxin reductase. Also the increase of glutathione peroxidase activities was substantially higher than that of thioredoxin reductase (400–1200% versus a maximum of 250%). These observations clearly indicate a catalytic role of the selenocysteine residue in the thioredoxin reductase, but suggest either the existence of a selenium‐unresponsive isoenzyme or a residual disulfide reductase activity in the selenium‐free truncated protein made under conditions of selenium deficiency. Show more

Keywords: Thioredoxin, thioredoxin reductase, selenium supplementation, tissue culture, selenocysteine residue, disulfide reductase activity

Citation: Biofactors, vol. 6, no. 3, pp. 351-358, 2012

Authors: , Omer Ch.A. | Anthony, N.J. | Buser‐Doepner, C.A. | Burkhardt, A.L. | deSolms, S.J. | Dinsmore, Ch.J. | Gibbs, J.B. | Hartman, G.D. | Koblan, K.S. | Lobell, R.B. | Oliff, A. | Williams, T.M. | Kohl, N.E.

Article Type: Research Article

Abstract: Ras, a signal‐transducing protein involved in mediating growth factor‐stimulated proliferation, is mutationally activated in over 30% of human tumors. To be functional Ras must bind to the inner surface of the plasma membrane, with post‐translational lipid modifications being necessary for this localization. The essential, first modification of Ras is farnesylation catalyzed by the enzyme farnesyl\, :\, proteintransferase (FPTase). Inhibitors of FPTase (FTIs) are currently being tested to determine if they are capable of tumor growth inhibition. Here we describe our efforts, along with those of other groups, in testing the biological and biochemical effects of FTIs.

Citation: Biofactors, vol. 6, no. 3, pp. 359-366, 2012

Authors: Fischer, Edmond H.

Article Type: Review Article

Citation: Biofactors, vol. 6, no. 3, pp. 367-374, 2012