Evidence for presence of a reduced form of digoxin-like immunoreactive factor (dihydro-DLIF) in mammalian tissues

1996 ◽  
Vol 42 (7) ◽  
pp. 1092-1099 ◽  
Author(s):  
H M Qazzaz ◽  
S A Jortani ◽  
J M Poole ◽  
R Valdes
Keyword(s):  
Lactone Ring ◽  
Fold Increase ◽  
Cross Reactivity ◽  
Reduced Form ◽  
Molar Ratio ◽  
Endogenous Ligand ◽  
Metabolic Route ◽  
Mammalian Tissues ◽  
Spectral Absorbance ◽  
Cytochrome P 450

Abstract Digoxin-like immunoreactive factor (DLIF) from adrenal glands is an endogenous ligand structurally related to the plant-derived cardiac glycoside digoxin. Cardiac glycosides regulate the activity of the sodium pump and thus play key roles in disease processes involving regulation of ion transport. We now report the discovery of an endogenous dihydro-DLIF analogous to dihydrodigoxin. We used HPLC, ultraviolet spectrophotometry, and cross-reactivity with two antibodies, one specific for digoxin and one for dihydrodigoxin, to support the hypothesis that dihydro-DLIF contains a chemically reduced lactone ring. The spectral absorbance maximum for dihydro-DLIF is at 196 nm, identical to dihydrodigoxin. DLIF and dihydro-DLIF are 975- and 2588-fold less immunoreactive than digoxin and dihydrodigoxin for their respective antibodies. The molar ratio of dihydro-DLIF to DLIF is approximately 5.3 in bovine adrenocortical tissue and approximately 0.38 in human serum. Dihydrodigoxin (reduced lactone ring) added to microsomes isolated from bovine adrenal cortex produced a 4.5-fold increase in digoxin-like immunoreactivity (oxidized lactone ring) after 3 h of incubation. The biotransformation is likely mediated by a cytochrome P-450 NADPH-dependent process. Our findings demonstrate the presence of a dihydro-DLIF in mammals and suggest a metabolic route for synthesis of endogenous DLIF in mammalian tissue.

scholarly journals Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique

1983 ◽  
Vol 211 (2) ◽  
pp. 333-340 ◽  
Author(s):  
E A Shephard ◽  
I R Phillips ◽  
R M Bayney ◽  
S F Pike ◽  
B R Rabin
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Fold Increase ◽  
Molar Ratio ◽  
Nadph Cytochrome ◽  
Specific Radioimmunoassay ◽  
Microsomal Membranes ◽  
Cytochrome P 450

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

scholarly journals IMMUNOHISTOCHEMICAL LOCALIZATION OF CATALASE IN MAMMALIAN TISSUES

1969 ◽  
Vol 17 (1) ◽  
pp. 30-35 ◽  
Author(s):  
SHIGERU MORIKAWA ◽  
TAKAYUKI HARADA
Keyword(s):  
Fluorescent Antibody ◽  
Bovine Liver ◽  
Cross Reactivity ◽  
Immunohistochemical Localization ◽  
Hepatic Cells ◽  
Surface Active Agents ◽  
Erythrocyte Catalase ◽  
Mammalian Tissues ◽  
Splenic Cells ◽  
Proximal Tubuli

The distribution of catalase was investigated in bovine tissues using fluorescent antibody techniques. The immunochemical properties of liver catalase were also examined. Two distinct components of liver catalase in immune system were found. Both of them possessed common antigenicity with erythrocyte catalase. No cross-reactivity was observed by immunodiffusion between catalase and the other hemoproteins such as lactoperoxidase, cytochrome c and hemoglobins. Bovine liver, pancreas, kidney, spleen and peripheral blood were examined. Catalase was located mainly in the cytoplasm of hepatic cells, acinar cells of the pancreas, epithelia of proximal tubuli, splenic cells scattered in the red pulp and some leukocytes. It was not found in any nucleus. Intracorpuscular catalase could be revealed in the erythrocytes treated with surface-active agents but not in frozen sections.

scholarly journals Conformational diversity facilitates antibody mutation trajectories and discrimination between foreign and self-antigens

2020 ◽  
Vol 117 (36) ◽  
pp. 22341-22350 ◽  
Author(s):  
Deborah L. Burnett ◽  
Peter Schofield ◽  
David B. Langley ◽  
Jennifer Jackson ◽  
Katherine Bourne ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Fold Increase ◽  
Cross Reactivity ◽  
Antibody Responses ◽  
X Ray ◽  
X Ray Crystallography ◽  
Conformational Diversity ◽  
High Discrimination ◽  
Complementarity Determining Regions ◽  
Self Antigen

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.

14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-α

2006 ◽  
Vol 290 (1) ◽  
pp. H55-H63 ◽  
Author(s):  
Xiang Fang ◽  
Shanming Hu ◽  
Bingkun Xu ◽  
Gary D. Snyder ◽  
Shawn Harmon ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Degradation Products ◽  
Expression System ◽  
Fold Increase ◽  
Peroxisome Proliferator ◽  
Binding Studies ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cell Expression ◽  
Cytochrome P 450 ◽  
Cell Expression System

Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-α (PPARα). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARα activator in a COS-7 cell expression system. Incubation with 10 μM 14,15-DHET produced a 12-fold increase in PPARα-mediated luciferase activity, an increase similar to that produced by the PPARα agonist Wy-14643 (20 μM). Although 10 μM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5( Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARα. However, PPARα was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPARα with a Kd of 1.4 μM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARα-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARα.

Cross Reactivity of Natural Hirudin Compared to the Recombinant Hirudins with Sheep Anti Hirudin Antibody

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4321-4321
Author(s):  
Vijaya L Paramatmuni ◽  
Debra Hoppensteadt ◽  
Saayi Setty ◽  
Evangelos Litinas ◽  
Nasir Sadeghi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Fold Increase ◽  
Cross Reactivity ◽  
Single Amino Acid ◽  
Scanning System ◽  
Recombinant Hirudin ◽  
Differential Behavior ◽  
Sds Page ◽  
Structural Differences ◽  
Antibody Complex

Abstract Abstract 4321 Recombinant versions of hirudin such as lepirudin (Refludan®) and desirudin (Iprivask®) are currently used as parenteral anticoagulants for various indications. The recombinant hirudin preparations differ from the natural hirudin in lacking a sulfate group on tyrosine at the 63rd position and a single amino acid substitution. It is hypothesized that these minor differences in the natural and recombinant versions of hirudins contribute to a differential immunogenic behavior. The purpose of this investigation was to compare the relative immunoreactivity of the three forms of hirudin to a sheep antin–hirudin antibody. Antibodies (anti-n-hirudin) were generated in sheep treated with natural hirudin isolated from the medicinal leech (Hirudo medicinalis ). SDS-PAGE immunoblot and Western transfer were conducted using the native hirudin and two recombinant hirudins against the sheep anti-n-hirudin IgG antibodies. Cross-reactivity was quantified by measuring total optical density of the bands using a UVP densitometric scanning system and chemiluminescence detection, by comparing the protein-antibody complex band density from the western blot showed a comparable behavior of the Desirudin and Lepirudin in contrast to the normal or native hirudin which exhibited a much higher band density (2-fold increase). Moreover, the native hirudin exhibited specific bands representing mono, di- and tetra-meric forms, whereas the two recombinant forms exhibited primarily monomeric and dimeric forms. Interestingly several other preclinical versions of recombinant hirudins exhibited fifferent immunoblotting patterns. Although, the structural differences and the molecular weight represent relatively minor variations in the natural and recombinant hirudins, these studies strongly suggest a differential behavior of natural and recombinant hirudins in terms of their cross-reactivity with anti-n-hirudin antibodies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Accelerated hepatic haem catabolism in the selenium-deficient rat

1977 ◽  
Vol 168 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R F Burk ◽  
M A Correia
Keyword(s):  
Urinary Excretion ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Haem Oxygenase ◽  
Cytochrome P 450

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.

scholarly journals Circadian Rhythm of Circulating Levels of the Endocannabinoid 2-Arachidonoylglycerol

2015 ◽  
Vol 100 (1) ◽  
pp. 220-226 ◽  
Author(s):  
E. C. Hanlon ◽  
E. Tasali ◽  
R. Leproult ◽  
K. L. Stuhr ◽  
E. Doncheck ◽  
...  
Keyword(s):  
Food Intake ◽  
Circadian Variation ◽  
Fold Increase ◽  
Structural Analog ◽  
Serum Concentrations ◽  
Endogenous Ligand ◽  
The Third ◽  
Lower Amplitude ◽  
Circulating Levels ◽  
Peripheral Metabolism

Abstract Context: The endocannabinoid (eCB) system is involved in the regulation of food intake and of peripheral metabolism. Although the cross talk between energy metabolism and the circadian system is well documented, little is known about a potential circadian modulation of human eCB activity. Objective: The objective of the study was to define the 24-hour profile of circulating levels of the most abundant endogenous ligand of the CB1 receptor, 2-arachidonoylglycerol (2-AG), in healthy young nonobese adults studied under controlled bedtime, dietary, and activity conditions. Methods: Fourteen subjects participated in this 4-day laboratory study with fixed light-dark cycles, standardized meals, and bedtimes. Sleep was recorded each night. On the third day, blood sampling at 15- to 30-minute intervals began at 9:30 pm and continued for 24 hours. Cortisol, leptin, and ghrelin were assayed on all samples, whereas the levels of 2-AG and its structural analog, 2-oleoylglycerol (2-OG), were measured at 60-minute intervals. Results: All participants exhibited a large circadian variation of 2-AG serum concentrations with a nadir around midsleep, coincident with the middle of the overnight fast. Levels of 2-AG increased continually across the morning, peaking in the early to midafternoon. Peak values represented, on average, a nearly 3-fold increase above nocturnal nadir levels. Concentrations of 2-OG followed a similar pattern, although with a shorter morning increase and lower amplitude. Conclusions: The findings demonstrate that activity of the eCB system is profoundly modulated by circadian rhythmicity and suggest that its impact on the regulation of food intake is suppressed during sleep and is maximal during early to midafternoon.

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