scholarly journals The effect of riboflavin deficiency on methylenetetrahydrofolate reductase (NADPH)(EC 1.5.1.20) and folate metabolism in the rat

1986 ◽  
Vol 55 (2) ◽  
pp. 455-464 ◽  
Author(s):  
C. J. Bates ◽  
N. J. Fuller
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Folate Metabolism ◽  
Hepatic Tissue ◽  
Riboflavin Deficiency ◽  
Weanling Rats ◽  
Metabolizing Enzyme ◽  
Riboflavin Deficient ◽  
Riboflavin Status

1. Riboflavin deficiency at two levels of severity was produced in weanling rats by feeding deficient diets for 6 weeks and using neck collars to prevent coprophagy. The severity of deficiency was monitored by growth, liver flavin levels and the activation coefficient of erythrocyte glutathione oxidoreductase (NAD(P)H) (EC 1. 6. 4. 2 ) Control groups, receiving the same diet with ample added riboflavin, were fed either ad lib., or were pair-fed with the deficient animals.2. The hepatic flavoenzyme, methylenetetrahydrofolate reductase (NADPH) (EC 1.5.1.20), was very markedly affected by severe riboflavin deficiency and was significantly, but less markedly, affected by the intermediate level of deficiency. This reduction in activity was due primarily to the direct effect of the diminished supply of riboflavin, and occurred to only a small extent as a result of inanition, demonstrated by a moderate reduction in activity in the more severely food-restricted of the two pair-fed groups. Since the enzyme is assayed in the presence of its flavin cofactor, FAD, it clearly cannot be reactivated in vitro, as some other depleted flavoenzymes can. The discriminatory ability in distinguishing between severe and moderate riboflavin deficiency in vivo confers some potential advantages on this oxidoreductase as a possible index of riboflavin status.3. The hepatic activity of another key folate-metabolizing enzyme, dihydrofolate reductase (EC 1.5.1.3), was not diminished by riboflavin deficiency in the present study.4. The ratio, labelled 5-methyltetrahydrofolic acid: other labelled compounds derived from intraperitoneally injected pteroylglutamic acid in extracts of hepatic tissue was significantly reduced in the riboflavin-deficient groups, indicating the possibility of an effect of riboflavin deficiency on folate metabolism in vivo.

scholarly journals Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Moe Ichikawa ◽  
Hiroki Akamine ◽  
Michika Murata ◽  
Sumito Ito ◽  
Kazuo Takayama ◽  
...  
Keyword(s):  
Small Intestine ◽  
Drug Absorption ◽  
Cell Model ◽  
Negative Effects ◽  
Piggybac Transposon ◽  
Drug Metabolizing Enzyme ◽  
Metabolizing Enzyme ◽  
Cyp3a4 Expression

AbstractCaco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

scholarly journals Protective Role of Coenzyme Q10 in Acute Sepsis-Induced Liver Injury in BALB/c Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Qian-wei Li ◽  
Qin Yang ◽  
Hong-Yang Liu ◽  
Yu-ling Wu ◽  
Yu-Hua Hao ◽  
...  
Keyword(s):  
Liver Injury ◽  
Coenzyme Q10 ◽  
Cecal Ligation ◽  
Protective Role ◽  
Hepatic Tissue ◽  
Control Group ◽  
Cecal Ligation And Puncture ◽  
Sequestosome 1

Sepsis increases the risk of the liver injury development. According to the research works, coenzyme Q10 exhibits hepatoprotective properties in vivo as well as in vitro. Current work aimed at investigating the protective impacts of coenzyme Q10 against liver injury in septic BALB/c mice. The male BALB/c mice were randomly segregated into 4 groups: the control group, the coenzyme Q10 treatment group, the puncture and cecal ligation group, and the coenzyme Q10+cecal ligation and puncture group. Cecal ligation and puncture was conducted after gavagaging the mice with coenzyme Q10 during two weeks. Following 48 h postcecal ligation and puncture, we estimated hepatic biochemical parameters and histopathological changes in hepatic tissue. We evaluated the expression of factors associated with autophagy, pyroptosis, and inflammation. Findings indicated that coenzyme Q10 decreased the plasma levels in alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase in the cecal ligation and puncture group. Coenzyme Q10 significantly inhibited the elevation of sequestosome-1, interleukin-1β, oligomerization domain-like receptor 3 and nucleotide-binding, interleukin-6, and tumor necrosis factor-α expression levels; coenzyme Q10 also increased beclin 1 levels. Coenzyme Q10 might be a significant agent in the treatment of liver injury induced by sepsis.

Exosomes derived from mmu_circ_0000623-modified ADSCs prevent liver fibrosis via activating autophagy

2020 ◽  
Vol 39 (12) ◽  
pp. 1619-1627 ◽  
Author(s):  
M Zhu ◽  
X Liu ◽  
W Li ◽  
L Wang
Keyword(s):  
Liver Fibrosis ◽  
Expression Profiles ◽  
Parenchymal Cell ◽  
Hepatic Tissue ◽  
Circular Rnas ◽  
In Vivo Experiments ◽  
Matrix Accumulation ◽  
Inhibitor Treatment

Prolonged parenchymal cell death leads to activation of fibrogenic cells, extracellular matrix accumulation, and eventually liver fibrosis. Increasing evidence shows that exosomes (Exos) secreted by adipose-derived mesenchymal stem cells (ADSCs) can be used to deliver circular RNAs (circRNAs) to treat liver fibrosis. To explore the uses of circRNA, circRNA expression profiles of hepatic tissue from normal and CCl4-induced mice were analyzed using high-throughput circRNA microarrays. The result showed that mmu_circ_0000623 expression was downregulated in CCl4-induced mice. Bioinformatics analysis and luciferin reporter experiments showed that mmu_circ_0000623 interacted with and regulated miR-125/ATG4D. In vitro and in vivo experiments showed that Exos from ADSCs, especially from mmu_circ_0000623-modified ADSCs, significantly suppressed CCl4-induced liver fibrosis by promoting autophagy activation. Autophagy inhibitor treatment significantly reversed the treatment effects of Exos. Proteins involved in autophagy and autophagy plaques positive for ATG4D expression were regulated by mmu_circ_0000623/miR-125. Our study found that Exos derived from mmu_circ_0000623-modified ADSCs prevented liver fibrosis via activating autophagy.

Pralatrexate Has Potent Activity Against Multiple Myeloma In Vitro and In Vivo, and Activity Correlates with Tumor RFC-1 and DHFR Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1831-1831 ◽  
Author(s):  
Michael Mangone ◽  
Luigi Scotto ◽  
Enrica Marchi ◽  
Owen A. O'Connor ◽  
Hearn J. Cho
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Research Funding ◽  
Folate Metabolism ◽  
Nog Mice ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Functional Biomarkers

Abstract Abstract 1831 Multiple myeloma (MM) is the second most common hematologic malignancy. Although there are effective new agents that can induce remission, relapse is inevitable and the disease is currently incurable. Progress in the treatment of this disease demands development of novel therapeutics and identification of functional biomarkers that may be used to distinguish tumors that are susceptible to specific targeted agents, creating a “personalized” therapeutic strategy for individual patients. We investigated these principles with anti-folates, which are not commonly used in MM but have demonstrated activity in this disease. Pralatrexate (PDX, 10-propargyl 10-deazaaminopterin) is a folate analogue that was rationally designed to have high affinity for Reduced Folate Carrier (RFC)-1, an oncofetal protein expressed in many cancers that actively transports folates into cells. PDX induced dose-dependent apoptotic cell death in a subset of human myeloma cell lines (HMCL) and CD138+ MM cells isolated from a clinical specimen. In sensitive cell lines, PDX exhibited 10-fold greater potency compared to the structurally related drug methotrexate (MTX). PDX induced dose-dependent, intrinsic apoptosis in sensitive HMCLs, characterized by cleavage of caspase-3 and -9 and accompanied by the loss of full-length Mcl-1, a Bcl-2 family protein that plays a critical role in drug-induced apoptosis in MM. Furthermore, the activity of PDX is not abrogated by the presence of exogenous interleukin-6 or by co-culture with HS-5 bone marrow stromal cells, both of which exert powerful survival effects on MM cells and can antagonize apoptosis in response to some cytotoxic chemotherapy drugs. Sensitivity to PDX-induced apoptosis correlated with higher relative levels of RFC-1 mRNA in sensitive compared to resistant HMCL. Resistant HMCL also exhibited a dose-dependent up-regulation of dihydrofolate reductase (DHFR) protein, a primary molecular target for anti-folates, in response to PDX exposure, whereas sensitive HMCL did not. These changes in functional folate metabolism biomarkers, high baseline RFC-1 expression and upregulation of DHFR in response to PDX, appeared to be mutually exclusive to sensitive or resistant HMCL, respectively. Importantly, PDX was also effective against sensitive HMCL in vivo in a novel mouse xenograft model. NOD/Shi-scid/IL-2Rγnull (NOG) mice were inoculated with MM.1s HMCL stably transduced to express both GFP and luciferase (GFP-luc). GFP-luc MM.1s cells engrafted into the long bones, pelvis, and vertebral column of NOG mice within 4–7 days after injection of cells, as assessed by in vivo bioluminescent imaging. Treatment with PDX resulted in a significant reduction in tumor burden after two doses. These results demonstrate that PDX has potent anti-myeloma activity in vitro and in vivo, and that RFC-1 expression and DHFR upregulation are robust functional biomarkers that may identify patients who are likely to benefit from PDX therapy. These data support further exploration of PDX therapy in clinical trials for MM and investigation of folate metabolism biomarkers as indices for treatment with this class of drugs. Improved anti-folates such as PDX are a promising class of agents that may be a valuable addition to the arsenal against MM. Disclosures: O'Connor: Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

scholarly journals Liberation of I4CO2from [14C]adipic acid and [14C]octanoic acid by adult rats during riboflavin deficiency and its reversal

1990 ◽  
Vol 63 (3) ◽  
pp. 553-562 ◽  
Author(s):  
C. J. Bates
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Adipic Acid ◽  
Time Course ◽  
Octanoic Acid ◽  
Urine Analysis ◽  
Acid Oxidation ◽  
Riboflavin Deficiency ◽  
Riboflavin Deficient

The purpose of the present study was to test the hypothesis that the already well-established mitochondria1 lesion in fatty acid oxidation in riboflavin-deficient experimental animals, might be accompanied by an alteration in vivo in the kinetics of oxidation of labelled adipic acid. This dicarboxylic acid was chosen for testing as a metabolic probe because a block in its oxidation was already apparent from urine analysis of riboflavin-deficient animals, whereas the oxidation of medium- or long-chain monocarboxylic acids seemed to be little affected by deficiency in vivo. Female adult Norwegian hooded rats fed on purified diets containing either 15 mg riboflavin/kg diet (controls) or about 0.4 mg/kg (riboflavin-deficient) received an intragastric dose of either [l,6-I4C] adipic acid or [l-'4C] octanoic acid. Expired carbon dioxide was then collected in an alkaline trap over 3 h, for determination of radioactivity.This test was repeated at intervals for up to 2 weeks following riboflavin repletion of the deficient animals, and in riboflavin-dosed controls. Whereas the rate and extent of ['4C]octanoic acid oxidation was not significantly affected by the deficiency or repletion, the extent of [I4C]adipic acid oxidation was markedly and significantly increased during repletion of the deficient animals. The time-course indicated a temporary overshoot, followed by a slow return to the control values over 1–2 weeks. Adipate oxidation was also much less affected by a preceding period of overnight starvation, than was octanoate oxidation. Thus, adipic acid (or a related metabolic probe) may have appropriate properties for the design of a functional test of fatty acid oxidation efficiency, during riboflavin deficiency or allied metabolic conditions in human subjects.

scholarly journals FMO1 catalyzes the production of taurine from hypotaurine

2019 ◽  
Author(s):  
Sunil Veeravalli ◽  
Ian R. Phillips ◽  
Rafael T. Freire ◽  
Dorsa Varshavi ◽  
Jeremy R. Everett ◽  
...  
Keyword(s):  
De Novo ◽  
Cysteic Acid ◽  
Muscle Disorders ◽  
H Nmr ◽  
Drug Metabolizing Enzyme ◽  
Anti Oxidant ◽  
Metabolizing Enzyme ◽  
Skeletal Muscle Disorders

ABSTRACTTaurine is one of the most abundant amino acids in mammalian tissues. It is obtained from the diet and by de novo synthesis, from cysteic acid or hypotaurine. Despite the discovery in 1954 that the oxygenation of hypotaurine produces taurine, the identification of an enzyme catalyzing this reaction has remained elusive. In large part this is due to the incorrect assignment, in 1962, of the enzyme as a NAD-dependent hypotaurine dehydrogenase. For more than 55 years the literature has continued to refer to this enzyme as such. Here we show, both in vivo and in vitro, that the enzyme that oxygenates hypotaurine to produce taurine is flavin-containing monooxygenase 1 (FMO1). Metabolite analysis of the urine of Fmo1-null mice by 1H NMR spectroscopy revealed a build-up of hypotaurine and a deficit of taurine in comparison with the concentrations of these compounds in the urine of wild-type mice. In vitro assays confirmed that FMO1 of human catalyzes the conversion of hypotaurine to taurine utilizing either NADPH or NADH as co-factor. FMO1 has a wide substrate range and is best known as a xenobiotic- or drug-metabolizing enzyme. The identification that the endogenous molecule hypotaurine is a substrate for the FMO1-catalyzed production of taurine resolves a long-standing mystery. This finding should help establish the role FMO1 plays in a range of biological processes in which taurine or its deficiency is implicated, including conjugation of bile acids, neurotransmitter, anti-oxidant and anti-inflammatory functions, the pathogenesis of obesity and skeletal muscle disorders.

scholarly journals Improvement Effect of Ficus vasculosa Ethanol Extract on D-galactose-Induced Mice Aging

2019 ◽  
Vol 14 (12) ◽  
pp. 1934578X1989667
Author(s):  
Jing-Jing Li ◽  
Ling Mo ◽  
Jia-Le Song
Keyword(s):  
Antioxidant Activity ◽  
Enzyme Activity ◽  
Reducing Power ◽  
Ethanol Extract ◽  
Trienoic Acid ◽  
Radical Scavenge Activity ◽  
Hepatic Tissue ◽  
Total Serum

This study was to investigate antioxidant activities of the ethanol extract from young edible leaves of Ficus vasculosa in vitro and in vivo . Ficus vasculosa ethanol extract (FVEE) showed significantly higher reducing power and α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenge activity than vitamin C ( P < 0.05). FVEE also showed an activity to resist the D-galactose-induced aging in mice assessed by serum and tissue levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). Total serum and tissue oxidative status, total antioxidantresponse, glutathione (GSH) and malondialdehyde (MDA) levels have been also measured. Pretreatment with FVEE at 200 mg/kg·body weight significantly increased enzyme activity of SOD and CAT in serum and hepatic tissue ( P < 0.05), as well as significantly increased enzyme activity of SOD in kidney ( P < 0.05). Furthermore, high concentration of FVEE pretreatment significantly increased the level of GSH in serum, hepatic tissue and kidney ( P < 0.05), meanwhile significantly decreased MDA production in hepatic tissue and kidney ( P < 0.05). In addition, the phytochemical investigation discovered six previously described compounds from FVEE, naringenin (1), vanillic acid (2), 9, 16-dioxo-10, 12, 14-octadeca-trienoic acid (3), 2, 6-dimethoxy-1, 4-benzoquinone (4), apigenin (5) and norartocarpetin (6), and all compounds were isolated from this plant for the first time. Among the various compounds found, the rare highly unsaturated fatty acid 9, 16-dioxo-10, 12, 14-octadeca-trienoic acid (3) has been identified, which had been isolated only once before from F. vasculosa. Evaluation of the antioxidant activity of isolated compounds showed naringenin (1) to be the most active. According to our research, FVEE present very high antioxidant activity in vitro due to the presence of several compounds known for their antioxidant activity such as flavonoid and phenolic acid. In vivo, the ethanol extract had improvement effects against D-galactose-induced aging by reducing oxidative stress.

scholarly journals Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro

2020 ◽  
Vol 21 (13) ◽  
pp. 4708
Author(s):  
Hannu Raunio ◽  
Olli Pentikäinen ◽  
Risto O. Juvonen
Keyword(s):  
Enzyme Activities ◽  
Biological Activities ◽  
Photophysical Properties ◽  
Fluorescent Sensors ◽  
Enzyme Assays ◽  
Highly Sensitive ◽  
Metabolizing Enzyme ◽  
Transfer Properties

in vivo methods, such as spectrophotometric, fluorometric, mass spectrometric,and radioactivity-based techniques. In fluorescence-based assays, the reaction produces a fluorescentproduct from a nonfluorescent substrate or vice versa. Fluorescence-based enzyme assays areusually highly sensitive and specific, allowing measurements on small specimens of tissues withlow enzyme activities. Fluorescence assays are also amenable to miniaturization of the reactionmixtures and can thus be done in high throughput. 7-Hydroxycoumarin and its derivatives arewidely used as fluorophores due to their desirable photophysical properties. They possess a large -conjugated system with electron-rich and charge transfer properties. This conjugated structure leadsto applications of 7-hydroxycoumarins as fluorescent sensors for biological activities. We describe inthis review historical highlights and current use of coumarins and their derivatives in evaluatingactivities of the major types of xenobiotic-metabolizing enzyme systems. Traditionally, coumarinsubstrates have been used to measure oxidative activities of cytochrome P450 (CYP) enzymes. For thispurpose, profluorescent coumarins are very sensitive, but generally lack selectivity for individual CYPforms. With the aid of molecular modeling, we have recently described several new coumarin-basedsubstrates for measuring activities of CYP and conjugating enzymes with improved selectivity.

Parathyroid hormone mRNA levels are increased by progestins and vary during rat estrous cycle

1996 ◽  
Vol 270 (1) ◽  
pp. E158-E163 ◽  
Author(s):  
E. Epstein ◽  
J. Silver ◽  
G. Almogi ◽  
N. Livni ◽  
T. Naveh-Many
Keyword(s):  
Parathyroid Hormone ◽  
Estrous Cycle ◽  
Primary Cultures ◽  
Parathyroid Tissue ◽  
Ovariectomized Rats ◽  
Mrna Levels ◽  
Physiological Relevance ◽  
Weanling Rats

Estrogen increases parathyroid hormone (PTH) mRNA levels in vivo in ovariectomized rats. We now show that the 19-norprogestin R-5020 given to weanling rats or mature ovariectomized rats led to a twofold increase in thyroparathyroid PTH mRNA levels. This increase in PTH mRNA occurred at 24 and 48 h after progesterone but not at 72 h. There were no changes in serum calcium. In vitro, in primary cultures of bovine parathyroid cells, progesterone increased PTH mRNA levels threefold at 10(-8) M and twofold at 10(-9) M after 24 h. Progesterone receptor (PR) mRNA was demonstrated in rat parathyroid tissue by in situ hybridization and in human parathyroid adenoma by immunohisto-chemistry. Changes in PTH mRNA levels during the rat estrous cycle were also studied. At proestrus and estrus PTH mRNA levels were increased significantly by three- and fourfold compared with diestrus. Our results confirm that the parathyroid gland is a target organ for the ovarian sex steroids estrogen and progesterone and are of physiological relevance as shown by the changes during estrus.

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