(1/34) The yeast A kinases differentially regulate iron uptake and respiratory function.
Yeast has three A kinase catalytic subunits, which have greater than 75% identity and are encoded by the TPK genes (TPK1, TPK2, and TPK3) [Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. (1987) Cell 50, 277-287]. Although they are redundant for viability, the three A kinases are not redundant for pseudohyphal growth [Robertson, L. S. & Fink, G. R. (1998) Proc. Natl. Acad. Sci. USA 95, 13783-13787; Pan, X. & Heitman, J. (1999) Mol. Cell. Biol. 19, 4874-4887]; Tpk2, but not Tpk1 or Tpk3, is required for pseudohyphal growth. Genome-wide transcriptional profiling has revealed unique signatures for each of the three A kinases leading to the identification of additional functional diversity among these proteins. Tpk2 negatively regulates genes involved in iron uptake and positively regulates genes involved in trehalose degradation and water homeostasis. Tpk1 is required for the derepression of branched chain amino acid biosynthesis genes that seem to have a second role in the maintenance of iron levels and DNA stability within mitochondria. The fact that TPK2 mutants grow better than wild types on nonfermentable carbon sources and on media deficient in iron supports the unique role of Tpk2 in respiratory growth and carbon source use. (+info)
(2/34) Antioxidant activity of Ferrozine-iron-amino acid complexes.
Amino acid-Fe(II)-chelator complexes exhibit strong antioxidant activity. Taking advantage of the unique spectral characteristics of the complexes formed when Ferrozine (Fz) is used as the chelator, we now show that the primary blue complex (epsilon(max) at 632 nm) decomposes by two independent pathways: (i) a nonoxidative pathway involving dissociation of the amino acid component and formation of a purple complex (epsilon(max) at 562 nm) and (ii) an oxidative pathway leading to Fe(III) and colorless products. Quantitative conversion of the blue to purple complex yields an isosbestic point (i.p.) at 601 nm, whereas no i.p. is formed during quantitative oxidation of the blue complex. However, under some experimental conditions, decomposition of the blue product occurs by both pathways, leading to occurrence of a clean i.p. at wavelengths varying from 601 to 574 nm. Results of simulation experiments, confirmed by direct analysis, demonstrate that shifts in the i.p. reflect differences in the fractions of blue compound that decompose by the oxidative and nonoxidative pathways. Indeed, the fraction of blue that is converted to the purple complex is readily deduced from the wavelength of the i.p. These results suggest that identification of a physiological chelator that can replace Ferrozine in amino acid-iron complexes might have important physiological and pharmacological applications. (+info)
(3/34) Centrifugal analyzer determination of ascorbate in serum or urine with Fe3+/Ferrozine.
Measurement of serum ascorbate may be useful in long-term population studies because of the possible influence of ascorbate on numerous physiological factors. We describe an automated method for determining ascorbate in serum and urine by using the reduction of ferric iron by ascorbate and the formation of a color between the resulting ferrous iron and Ferrozine [3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine]. A centrifugal analyzer is used to rapidly and simultaneously measure ascorbate in the samples and standards and minimize interference from slower reacting substances in the sample. The method is highly precise and specific. Data are also presented on the stability of ascorbate in serum, urine, and aqueous solutions. (+info)
(4/34) Spuriously low concentrations of serum iron measured with generation 14 Kodak Ektachem slides: prevalence, possible causes, and partial improvement with generation 16 slides.
For sera with iron (Fe) concentrations < 4 mumol/L, Kodak Ektachem slides Generation (GEN) 14 (without ascorbic acid) yielded systematically lower results for Fe than did liquid Ferrozine-based reagents from Baker containing ascorbic acid (10 g/L, final concentration) and adapted to Cobas-Bio. During an 8-month comparison period, outliers (defined as [Fe]Cobas - [Fe]Kodak > 4 mumol/L) were seen in 21 of the 8731 sera (0.24%) tested, corresponding to < 5% of the sera with [Fe]Kodak < 4 mumol/L. In vitro addition of ascorbic acid and (or) Fe identified at least two types of outliers: type 1 (approximately 70%), characterized by [Fe]Kodak > 0.4 mumol/L, by (supra)normal Fe recovery in Kodak slides in the presence or absence of ascorbic acid (10 g/L), and by between-method differences in serum Fe (Cobas - Kodak) that were significantly correlated with serum Zn content (P < 0.0004); and type 2 (approximately 30%), tentatively ascribed to contamination by EDTA, with serum Fe by Kodak < 0.4 mumol/L and Fe recovery near 0%, both of which could be significantly and dose-dependently increased by addition of ascorbic acid (5-20 g/L). For both types of outliers, flameless atomic absorption spectrometry (AAS) yielded results that were significantly higher than concentrations by Kodak with GEN 14. Use of GEN 16 slides (containing ascorbic acid) improved concordance of Kodak results with Cobas, and hence with flameless AAS, for both types of outliers; abolished Zn dependency of results; and increased Fe results in sera with type 2 outliers, although these remained substantially lower than by Cobas. However, like other ascorbic acid-containing reagents, GEN 16 slides were more sensitive to interference by dextran-bound Fe, as assessed during in vitro addition experiments and comparisons involving samples from Fe-dextran-treated patients. GEN 16 slides are hence expected to more frequently overestimate the physiologically available protein-bound Fe in hemodialysis patients. In hospital laboratories, this new interference will probably arise more frequently than the spuriously low results with GEN 14, hence warranting further efforts in optimizing Fe slides. (+info)
(5/34) Iron and 8-isoprostane levels in acute and chronic wounds.
The purpose of this study was to determine differences in iron and iron protein (ferritin and transferrin) levels in chronic venous ulcers and acute wounds. The deleterious effect of iron in free-radical-induced tissue damage was indirectly examined by assessing 8-isoprostane levels and antioxidant status in wound fluid samples. Wound fluid samples from chronic leg ulcers in nonhealing and healing phases and wound fluid from mastectomy wounds were assayed for ferritin, transferrin, total iron, 8-isoprostane, and total antioxidant status. Immunohistochemistry and Perls' staining were performed on paired biopsies from chronic leg ulcers and on normal skin biopsies. Chronic wound fluid had significantly greater levels of ferritin (p < 0.05) and lower levels of transferrin (p < 0.001) than acute wound fluid and there was a significant reduction in the level of ferritin in healing compared to nonhealing chronic leg ulcers (p < 0.05). No significant differences were observed in the levels of total iron present in the wound fluids. Histologic staining showed consistently more ferritin and ferric iron in chronic wound tissue than in normal skin. Elevated levels of 8-isoprostane and antioxidants were observed for chronic wound fluid compared to acute wound fluid (p < 0.001). These results suggest the existence of an environment of oxidative stress in chronic wounds and the likely contribution of iron to exacerbating tissue damage and delaying healing in these wounds. (+info)
(6/34) Colorimetric measurement of iron in plasma samples anticoagulated with EDTA.
AIMS: To determine if the iron in EDTA anticoagulated plasma samples can be measured by colorimetric assays using Ferrozine. METHODS: Paired samples of serum and EDTA plasma were obtained from 24 patients and analysed by three commercial iron methods. The EDTA plasmas were also analysed using methods modified by the addition of zinc sulphate or with different concentrations of Ferrozine. The iron contamination of EDTA sample tubes was measured by atomic absorption spectroscopy. RESULTS: Two commercial colorimetric iron methods gave results of zero for EDTA plasma samples. A third commercial reagent gave plasma results that were about 30% lower than their corresponding serum samples. Addition of 7 mmol/l zinc sulphate to this reagent system and extending the sample preincubation time to 300 seconds yielded comparable results from paired serum and EDTA plasma samples. Linear regression analysis gave a slope of 0.97 with an intercept of 0.60 mumol/l and R2 = 0.9943. Measurements by atomic absorption spectroscopy showed that this positive intercept was due to contamination of the blood collection tubes with about 90 ng of iron. CONCLUSIONS: Modification of commercial colorimetric iron methods permits the biochemical assessment of iron status and a full blood count from a single EDTA anticoagulated blood sample. (+info)
(7/34) Elimination of fibrinogen interference in a dye-binding method for iron.
Serum iron concentrations greater than 90 mumol/L were measured in samples from hemodialysis patients by a Ferrozine dye-binding method. Reanalysis by coulometry showed these results to be spuriously high. Turbidity resulting from precipitation of fibrinogen was identified as the source of the interference. High concentrations of heparin enhanced the effect. We conclude that the persistence of fibrinogen in serum samples from patients treated with anticoagulants is a potential interference in Ferrozine dye-binding methods performed without prior deproteinization. Adding guanidine.HCl to the acid buffer reagent is a simple way to eliminate this interference. (+info)
(8/34) Mouse liver cytidine-5'-monophosphate-N-acetylneuraminic acid hydroxylase. Catalytic function and regulation.
In this paper, we present the results of an investigation into the catalytic properties of CMP-Neu5Ac hydroxylase (Neu5Ac: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory. The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-Neu5Ac hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-Neu5Gc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar-nucleotides. Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome b5 and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase. Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome b5 may thus serve as an electron donor for this postulated cofactor. (+info)