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1.
Sci Rep ; 5: 13222, 2015 Sep 23.
Article in English | MEDLINE | ID: mdl-26394756

ABSTRACT

Strong evidence exists for a central role of amyloid ß-protein (Aß) oligomers in the pathogenesis of Alzheimer's disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aß aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAß3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aß aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.


Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/metabolism , Protein Aggregation, Pathological/drug therapy , Animals , Carrier Proteins/pharmacology , Catechin/analogs & derivatives , Catechin/pharmacology , Disease Models, Animal , Ferredoxin-NADP Reductase/pharmacology , Humans , Inositol/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Oligopeptides/pharmacology , Taurine/analogs & derivatives , Taurine/pharmacology
2.
Arch Environ Contam Toxicol ; 44(1): 43-52, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12434218

ABSTRACT

A selected suite of cytochemical parameters in Mytilus edulis are altered in response to field and laboratory exposure to chemical contaminants. These biomarkers include lysosomal stability, nicotinamide adenine dinucleotide phosphate (NADPH)-ferrihemoprotein reductase activity, liposfuscin deposition, and accumulation of lysosomal and cytoplasmic unsaturated neutral lipid. Normal variations in physiological processes (influenced by exogenous seasonal changes in temperature, salinity, food availability, etc.) may alter the sensitivity of these biomarkers to contaminant exposure. To address this issue, M. edulis (complex) were sampled monthly from a reference nonurban site (Coupeville, Penn Cove) and a polluted urban site (Seacrest, Elliott Bay) in Puget Sound, WA, for a period of 15 months. Physiological measurements including total length, total weight, somatic and mantle weights (an indication of gonadal development and reproductive status), condition index, and the presence or absence of hemic neoplasia (HN, or leukemia) were recorded. Significant differences in lysosomal stability, lysosomal and cytoplasmic unsaturated neutral lipids, lipofuscin deposition, and NADPH-ferrihemoprotein reductase activity in cells of the digestive gland or digestive tubules were generally found in mussels taken throughout the year from Seacrest compared to mussels sampled from Coupeville, consistent with exposure to chemical contaminants. No seasonally influenced suppression of the entire suite of parameters as measures of contaminant exposure was evident. Therefore these biomarkers can be used to evaluate contaminant exposure in mussels throughout the entire year.


Subject(s)
Adaptation, Physiological , Biomarkers/analysis , Bivalvia/physiology , Water Pollutants/adverse effects , Animals , Body Weight , Environmental Monitoring , Ferredoxin-NADP Reductase/analysis , Ferredoxin-NADP Reductase/pharmacology , Lipid Metabolism , Lipofuscin/metabolism , Lysosomes/metabolism , Reproduction , Seasons
3.
Eur J Biochem ; 263(2): 319-25, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10406938

ABSTRACT

We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem. 244, 835-839]. The aim of the present study was to determine whether it is adrenodoxin reductase, adrenodoxin or both which limits cytochrome P450scc activity and hence progesterone synthesis in the placenta. We found that the concentrations of adrenodoxin reductase and adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous adrenodoxin reductase to sonicated mitochondria rescued pregnenolone synthesis to a level above that for intact mitochondria, showing that adrenodoxin is near-saturating in vivo. In contrast, pregnenolone synthesis by sonicated mitochondria was almost zero even after the addition of human adrenodoxin. This shows that the concentration of endogenous adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine adrenodoxin reductase have revealed that a twofold higher concentration of human adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human adrenodoxin. Thus, not only is the adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of progesterone synthesis.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/antagonists & inhibitors , Cytochrome P-450 Enzyme System/metabolism , Ferredoxin-NADP Reductase/pharmacology , Placenta/drug effects , Placenta/enzymology , Animals , Cattle , Dose-Response Relationship, Drug , Ferredoxin-NADP Reductase/analysis , Ferredoxin-NADP Reductase/isolation & purification , Humans , Kinetics , Mitochondria/enzymology , Pregnenolone/biosynthesis
4.
Arch Biochem Biophys ; 363(2): 373-6, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10068461

ABSTRACT

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins.


Subject(s)
Mitochondria/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Adrenodoxin/genetics , Adrenodoxin/pharmacology , Apoproteins/metabolism , Biological Transport , Cell Division/drug effects , Cell-Free System , Cholesterol Side-Chain Cleavage Enzyme/metabolism , Ferredoxin-NADP Reductase/genetics , Ferredoxin-NADP Reductase/pharmacology , Fungal Proteins/metabolism , Mitochondria/drug effects , Mitochondrial Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Saccharomyces cerevisiae
5.
Eur J Biochem ; 244(3): 835-9, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9108254

ABSTRACT

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P-450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P-450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3-fold higher than that for intact mitochondria. Similar results were observed when 20alpha-hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P-450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P-450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/metabolism , Cholesterol/metabolism , Placenta/metabolism , Adrenodoxin/metabolism , Adrenodoxin/pharmacology , Animals , Cattle , Electron Transport/drug effects , Female , Ferredoxin-NADP Reductase/metabolism , Ferredoxin-NADP Reductase/pharmacology , Humans , In Vitro Techniques , Mitochondria/drug effects , Mitochondria/metabolism , Placenta/drug effects , Pregnancy , Pregnenolone/biosynthesis , Rats , Sonication , Swine
7.
Eur J Biochem ; 51(1): 145-54, 1975 Feb 03.
Article in English | MEDLINE | ID: mdl-235426

ABSTRACT

18-Hydroxylation of deoxycorticosterone was studies with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P450 reductase preparation from bovine adrenals. In some experimenta a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-45o reductase was to some extent rate-limiting. The rate of 18-hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rat of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11beta-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11beta- and 18hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11beta- and 18-hydroxylation of deoxycorticosterone.


Subject(s)
Adrenal Glands/metabolism , Desoxycorticosterone/metabolism , Adrenodoxin/pharmacology , Animals , Cattle , Cytochrome P-450 Enzyme System/pharmacology , Cytochrome Reductases/pharmacology , Ferredoxin-NADP Reductase/pharmacology , Hydroxylation , Male , Mitochondria/metabolism , NADP/pharmacology , Rats , Species Specificity , Steroids/pharmacology
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