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Sample records for 3-hydroxy-3-methylglutaryl coa reductase

  1. Enhancement of cardenolide and phytosterol levels by expression of an N-terminally truncated 3-hydroxy-3-methylglutaryl CoA reductase in Transgenic digitalis minor.

    PubMed

    Sales, Ester; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2007-06-01

    Pathway engineering in medicinal plants attains a special significance in Digitalis species, the main industrial source of cardiac glycosides, steroidal metabolites derived from mevalonic acid via the triterpenoid pathway. In this work, the Arabidopsis thaliana HMG1 cDNA, coding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the MVA pathway, was expressed in the cardenolide-producing plant Digitalis minor. Transgenic plants were morphologically indistinguishable from control wild plants and displayed the same developmental pattern. Constitutive expression of HMG1 resulted in an increased sterol and cardenolide production in both in vitro- and greenhouse-grown plants. This work demonstrates that transgenic D. minor plants are a valuable system to study and achieve metabolic engineering of the cardenolide pathway and in consequence for the genetic improvement of Digitalis species.

  2. Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia.

    PubMed

    Muñoz-Bertomeu, Jesús; Sales, Ester; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2007-11-01

    Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, beta-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T1 plants derived from self-pollinated seeds of T0 lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.

  3. Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Yu, Xiaohong; Hao, Xiaolong; Huang, Qiang; Xiao, Jianbo; Cui, Lijie; Kai, Guoyin

    2014-09-01

    Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.

  4. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  5. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed Central

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control. PMID:10861207

  6. Short- and long-term regulation of 3-hydroxy 3-methylglutaryl coenzyme A reductase by a 4-methylcoumarin.

    PubMed

    Trapani, Laura; Segatto, Marco; Simeoni, Veronica; Balducci, Valentina; Dhawan, Ashish; Parmar, Virinder S; Prasad, Ashok K; Saso, Luciano; Incerpi, Sandra; Pallottini, Valentina

    2011-07-01

    Dyslipidemia is one of the most significant risk factors for cardiovascular diseases. Cholesterol homeostasis is regulated by both the receptor-mediated endocytosis of Low Density Lipoproteins by LDL receptors and de novo cholesterol synthesis via the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. Although statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase substrate competitors, have revolutionized the management of cardiovascular diseases by lowering serum LDL, their side effects range from myalgia to rhabdomyolysis. Treatment with antioxidant compounds could represent an efficient alternative in the modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Indeed it has already been demonstrated that the rise in reactive oxygen species levels causes the complete dephosphorylation and, in turn activation of the enzyme. Many coumarins and their derivatives have the special ability to scavenge reactive oxygen species or show a lipid lowering potential. Here we evaluated whether the coumarin, 4-methylesculetin could exert both the ability to scavenge ROS and to modulate 3-hydroxy-3-methylglutaryl coenzyme A reductase in HepG2 cell line where the enzyme activity dysregulation induced by reactive oxygen species has already been reported. The antioxidant property of 4-methylesculetin led to the reduction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activation state through the increase of the enzyme phosphorylation. In addition, this coumarin showed the ability to modulate 3-hydroxy-3-methylglutaryl coenzyme A reductase protein levels both by transcriptional and degradational events independent of its antioxidant activity.

  7. Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

    PubMed Central

    Aoyagi, K; Beyou, A; Moon, K; Fang, L; Ulrich, T

    1993-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat. PMID:8108513

  8. One statin, two statins, three statins, more: similarities and differences of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

    PubMed

    Turkoski, Beatrice B

    2011-01-01

    Statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are one of the most widely prescribed drugs today. They are considered first-line therapy to lower blood serum cholesterol levels in conjunction with therapeutic lifestyle changes for both primary and secondary prevention of cardiovascular events. In the following discussion, a brief explanation of the background of statins will explain why they are deemed so important today. The similarities and differences between the different statins will be addressed, including a look at dosage, side effects, and cautions for the seven 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors currently available.

  9. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

    PubMed Central

    Dimster-Denk, D; Thorsness, M K; Rine, J

    1994-01-01

    In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation. Images PMID:7949422

  10. 3-hydroxy 3-methylglutaryl coenzyme A reductase inhibition impairs muscle regeneration.

    PubMed

    Trapani, Laura; Segatto, Marco; La Rosa, Piergiorgio; Fanelli, Francesca; Moreno, Sandra; Marino, Maria; Pallottini, Valentina

    2012-06-01

    Skeletal muscle has the ability to regenerate new muscle fibers after injury. The process of new muscle formation requires that quiescent mononuclear muscle precursor cells (myoblasts) become activated, proliferate, differentiate, and fuse into multinucleated myotubes which, in turn, undergo further differentiation and mature to form functional muscle fibers. Previous data demonstrated the crucial role played by 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthetic pathway, in fetal rat myoblast (L6) differentiation. This finding, along with epidemiological studies assessing the myotoxic effect of statins, HMGR inhibitors, allowed us to speculate that HMGR could be strongly involved in skeletal muscle repair. Thus, our research was aimed at evaluating such involvement: in vitro and in vivo experiments were performed on both mouse adult satellite cell derived myoblasts (SCDM) and mouse muscles injured with cardiotoxin. Results demonstrate that HMGR inhibition by the statin Simvastatin reduces SCDM fusion index, fast MHC protein levels by 60% and slow MHC by 40%. Most importantly, HMGR inhibition delays skeletal muscle regeneration in vivo. Thus, besides complaining of myopathies, patients given Simvastatin could also undergo an impairment in muscle repair.

  11. Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase.

    PubMed Central

    Campos, N; Boronat, A

    1995-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate. This is the first committed step of isoprenoid biosynthesis. A common feature of all known plant HMGR isoforms is the presence of two highly conserved hydrophobic sequences in the N-terminal quarter of the protein. Using an in vitro system, we showed that the two hydrophobic sequences of Arabidopsis HMGR1S function as internal signal sequences. Specific recognition of these sequences by the signal recognition particle mediates the targeting of the protein to microsomes derived from the endoplasmic reticulum. Arabidopsis HMGR is inserted into the microsomal membrane, and the two hydrophobic sequences become membrane-spanning segments. The N-terminal end and the C-terminal catalytic domain of Arabidopsis HMGR are positioned on the cytosolic side of the membrane, whereas only a short hydrophilic sequence is exposed to the lumen. Our results suggest that the plant HMGR isoforms known to date are primarily targeted to the endoplasmic reticulum and have the same topology in the membrane. This reinforces the hypothesis that mevalonate is synthesized only in the cytosol. The possibility that plant HMGRs might be located in different regions of the endomembrane system is discussed. PMID:8718626

  12. Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Leivar, Pablo; González, Víctor M; Castel, Susanna; Trelease, Richard N; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of

  13. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

    PubMed Central

    Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR

  14. Identification of a 3-hydroxy-3-methylglutaryl-CoA reductase gene highly expressed in the root tissue of Taraxacum kok-saghyz

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Kazak dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) en...

  15. Robinetinidol-flavone attenuates cholesterol synthesis in hepatoma cells via inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Niu, Hai; Chao, Yu; Li, Ke; Li, Junxiang; Gong, Weihong; Huang, Wen

    2015-01-01

    Robinetinidol-(4β,2')-tetrahydroxy-flavone (RBF) is an oligomeric condensed polyphenol that has been shown to exhibit anti-obesity effects in mice. However, little is know regarding its effect on cholesterol synthesis. The present study therefore aimed to investigate the effect of RBF on cholesterol synthesis. It was determined that RBF decreased serum total cholesterol and low density lipoprotein cholesterol in rats by 25.9 and 50.8%, respectively (P<0.001). These results strengthen evidence for the hypothesis that RBF exerts anti-atherogenic effects in vivo. Furthermore, RBF decreased cholesterol synthesis by 72%, when measured using a 3 h period of radiolabeled acetate incorporation into cholesterol, but not when using radiolabelled mevalonate, suggesting that RBF-mediated inhibition occurred largely at or above the level of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase. The mechanism by which RBF inactivates HMG-CoA reductase may be attributed to the induction of phosphorylation of adenosine monophosphate (AMP)-kinase, since these results showed that RBF increased phosphorylation of AMP-kinase and HMG-CoA reductase by 2.1- and 3.2-fold, respectively, within 30 min of addition. These results suggest that RBF may be a potential therapeutic agent for hypercholesteremia.

  16. Charaterization of bumarsin, a 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitor from Mesobuthus martensii Karsch venom.

    PubMed

    Chai, S C; Armugam, A; Strong, P N; Jeyaseelan, K

    2012-09-01

    Scorpion venoms are rich sources of bioactive peptides and are widely known for their ion channel inhibiting properties. We have isolated, cloned and characterized a venom protein (Bumarsin) from the Chinese scorpion, Mesobuthus martensii Karsch. Bumarsin cDNA encodes a 8132 Da, 72 amino acid mature protein that most probably exists in its native form as a Cys-bridged homodimer. We have identified this novel protein to be an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. 0.6 μM of Bumarsin inhibits 32% of the HMG-CoA reductase activity, in comparison to 10 μM simvastatin which only inhibits 35% of the activity. RT-PCR and SELDI-TOF mass spectrometric studies demonstrate that bumarsin regulates the expression of both genes and proteins involved in cholesterol homeostasis. Our results suggest that bumarsin may provide a model for the design of novel drugs that can be used to modulate cholesterol homeostasis.

  17. Thermodynamic and Structure Guided Design of Statin Based Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

    SciTech Connect

    Sarver, Ronald W.; Bills, Elizabeth; Bolton, Gary; Bratton, Larry D.; Caspers, Nicole L.; Dunbar, James B.; Harris, Melissa S.; Hutchings, Richard H.; Kennedy, Robert M.; Larsen, Scott D.; Pavlovsky, Alexander; Pfefferkorn, Jeffrey A.; Bainbridge, Graeme

    2008-10-02

    Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2--7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC{sub 50} = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a {Delta}H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.

  18. Thermodynamic and structure guided design of statin based inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

    PubMed

    Sarver, Ronald W; Bills, Elizabeth; Bolton, Gary; Bratton, Larry D; Caspers, Nicole L; Dunbar, James B; Harris, Melissa S; Hutchings, Richard H; Kennedy, Robert M; Larsen, Scott D; Pavlovsky, Alexander; Pfefferkorn, Jeffrey A; Bainbridge, Graeme

    2008-07-10

    Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2-7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC 50 = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a Delta H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.

  19. Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

    PubMed Central

    Luessi, Felix; Bendix, Ivo; Paterka, Magdalena; Prozorovski, Timour; Treue, Denise; Luenstedt, Sarah; Herz, Josephine; Siffrin, Volker; Infante-Duarte, Carmen; Zipp, Frauke; Waiczies, Sonia

    2014-01-01

    The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities. PMID:25013913

  20. Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues.

    PubMed

    Kondo, Katsuyoshi; Uritani, Ikuzo; Oba, Kazuko

    2003-05-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.

  1. Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

    PubMed Central

    Song, Adelene Ai-Lian; Abdullah, Janna Ong; Abdullah, Mohd. Puad; Shafee, Norazizah; Othman, Roohaida; Tan, Ee-Fun; Noor, Normah Mohd.; Raha, Abdul Rahim

    2012-01-01

    Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction. PMID:23300671

  2. Vanadium and ascorbate effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol and tissue minerals in guinea pigs fed low-chromium diets.

    PubMed

    Seaborn, C D; Mitchell, E D; Stoecker, B J

    Vanadium has been reported to affect numerous physiological processes; however, a demonstration that vanadium deficiency consistently impairs biological function is lacking. The purpose of this study was to determine if the activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, is affected by dietary supplementation of vanadate and/or chronic ascorbic acid deficiency. To determine if vanadium and/or ascorbic acid affected mineral metabolism, tissue minerals also were analyzed. Weanling male guinea pigs were assigned randomly to groups of 10 in a 2 x 2 factorial design. The dietary variables were ascorbate, 0.5 or 10 mg/day, and vanadium < 0.01 microgram or 0.5 microgram/g diet as NH4VO3 in a low Cr diet containing < 0.07 microgram Cr/g diet. After 21 weeks on this diet, guinea pigs receiving more ascorbate had lower liver weight/body weight ratios and increased bone copper. Testes weight/body weight ratios, hepatic glycogen and bone copper decreased while hepatic lipids, fecal bile acids, plasma cortisol and bone calcium and magnesium were increased by vanadium supplementation. An interaction between vanadium and ascorbate affected cholesterol excretion in feces, hepatic iron, plasma cholesterol concentration and the activity of HMG CoA reductase. This study provides evidence of increased bone mineral concentrations with vanadium supplementation and of an interaction between vanadium and ascorbate which affected cholesterol metabolism.

  3. Circadian variation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in swine liver and ileum.

    PubMed

    Rogers, D H; Kim, D N; Lee, K T; Reiner, J M; Thomas, W A

    1981-07-01

    The temporal variation of HMG-CoA reductase activity in the liver and intestine of swine was investigated. The thin-layer chromatographic method widely used in the assay of the reductase was successfully applied to the porcine enzymes. Parallel circadian rhythms were demonstrated in both hepatic and ileal reductases from mash-fed animals. Peak activity occurred approximately 6 hr after feeding, 2.7-fold over the basal level in the liver, and 1.6-fold in the ileum. A milk-cholesterol diet caused a marked depression of both rhythms (90% in liver, 50% in ileum); however, the hourly variation in activity persisted in both organs. Cholestyramine was found to elevate hepatic activity (2.7-fold throughout the rhythm) without affecting that of the intestine. Clofibrate had no effect on either enzyme at any time during the cycle despite a 34% reduction in serum cholesterol concentrations.

  4. Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers

    SciTech Connect

    Roth, M.; Emmons, L.R.; Perruchoud, A. ); Block, L.H. )

    1991-03-01

    The plausible role that platelet-derived growth factor (PDGF) has in the localized pathophysiological changes that occur in the arterial wall during development of atherosclerotic lesions led the authors to investigate the influence of recombinant (r)PDGF isomers -AA, -AB, and -BB on the expression of low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG0CoA) reductase ((S)-mevalonate:NAD{sup +} oxidoreductase (CoA-acylating), EC 1.1.1.88) genes. In addition, they clarified the role of protein kinase C (PKC) in expression of the two genes in human skin fibroblasts and vascular smooth muscle cells. The various rPDGF isoforms are distinct in their ability to activate transcription of both genes: (i) both rPDGF-AA and -BB stimulate transcription of the LDL-R gene; in contrast, rPDGF-BB but not -AA, activates transcription of the HMG-CoA reductase gene; (ii) all recombinant isoforms of PDGF activate transcription of the c-fos gene; (iii) while rPDGF-dependent transcription of the lDL-R gene occurs independently of PKC, transcription of the HMG-CoA reductase gene appears to involve the action of that enzyme.

  5. In vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, pravastatin, lovastatin, and simvastatin, using neonatal rat skeletal myocytes.

    PubMed

    Masters, B A; Palmoski, M J; Flint, O P; Gregg, R E; Wang-Iverson, D; Durham, S K

    1995-03-01

    Pravastatin, lovastatin, and simvastatin, drugs which lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, have been linked to skeletal myopathies in humans and rats. The myotoxicity of these three drugs was compared, after 48 hr exposure, in cultures of primary neonatal rat skeletal myotubes. Measurements included HMG CoA reductase activity ([14C]acetate incorporation into cholesterol), indicators of membrane damage (CPK, LDH, and AST), cell viability (mitochondrial dehydrogenase metabolism of MTT), protein synthesis ([3H]leucine incorporation), and energy status (ATP). All three drugs inhibited cholesterol synthesis to the same extent in rat hepatocytes (IC50s approximately 0.07 microM). Lovastatin- and simvastatin-induced inhibition of cholesterol synthesis in myotubes was unchanged compared to that of hepatocytes, but pravastatin was 85-fold less potent (IC50 = 5.9 microM). Protein synthesis and ATP levels were the most sensitive indicators of toxicity. Pravastatin (IC50 = 759 microM) was > 100-fold less inhibitory of protein synthesis than lovastatin (IC50 = 5.4 microM) or simvastatin (IC50 = 1.9 microM). Addition of mevalonic acid (the immediate product of the HMG CoA reductase reaction), as 100 microM mevalonic acid lactone, reversed the toxicity of all three drugs. Removal of serum for 24-72 hr did not alter the toxicity of any of the drugs compared to cultures containing 10% serum, suggesting that differences in protein binding did not account for the differences in toxicity of the drugs. These results indicate that pravastatin is less myotoxic than lovastatin or simvastatin in this in vitro system using neonatal rat skeletal muscle cells, and this differential toxicity is correlated with the selective decrease in inhibition of HMG CoA reductase by pravastatin in nonhepatic tissues.

  6. Metabolism and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in transplant patients: are the statins mechanistically similar?

    PubMed

    Christians, U; Jacobsen, W; Floren, L C

    1998-10-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) inhibitors are the most effective drugs to lower cholesterol in transplant patients. However, immunosuppressants and several other drugs used after organ transplantation are cytochrome P4503A (CYP3A, EC 1.14.14.1) substrates. Pharmacokinetic interaction with some of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, specifically lovastatin and simvastatin, leads to an increased incidence of muscle skeletal toxicity in transplant patients. It is our objective to review the role of drug metabolism and drug interactions of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and cerivastatin. In the treatment of transplant patients, from a drug interaction perspective, pravastatin, which is not significantly metabolized by CYP enzymes, and fluvastatin, presumably a CYP2C9 substrate, compare favorably with the other statins for which the major metabolic pathways are catalyzed by CYP3A.

  7. Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A[W

    PubMed Central

    Leivar, Pablo; Antolín-Llovera, Meritxell; Ferrero, Sergi; Closa, Marta; Arró, Montserrat; Ferrer, Albert; Boronat, Albert; Campos, Narciso

    2011-01-01

    Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B′′ regulatory subunits of protein phosphatase 2A (PP2A), designated B′′α and B′′β, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B′′α and B′′β are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B′′α and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B′′β. Our data indicate that PP2A exerts multilevel control on HMGR through the five-member B′′ protein family during normal development and in response to a variety of stress conditions. PMID:21478440

  8. Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens.

    PubMed

    Mosley, S T; Kalinowski, S S; Schafer, B L; Tanaka, R D

    1989-09-01

    Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was

  9. Brain and Optic System Pathology in Hypocholesterolemic Dogs Treated with a Competitive Inhibitor of 3-hydroxy-3-methylglutaryl Coenzyme A Reductase

    PubMed Central

    Berry, P. H.; MacDonald, J. S.; Alberts, A. W.; Molon-Noblot, S.; Chen, J. S.; Lo, C.-Y. L.; Greenspan, M. D.; Allen, H.; Durand-Cavagna, G.; Jensen, R.; Bailly, Y.; Delort, P.; Duprat, P.

    1988-01-01

    The cholesterol lowering compound lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34 HMG CoA reductase), was given in nine separate experiments to normocholesterolemic dogs at rates up to 180 times the maximum therapeutic dose in man (1 mg/kg/day). Mean serum total cholesterol concentrations were reduced as much as 88% below normal. Clinical evidence of neurotoxicity occurred in up to 37% of animals given 180 mg/kg/day lovastatin for 11 or more days, especially in one laboratory where the dosing regime resulted in higher concentrations of plasma drug levels. Dogs receiving 60 mg/kg/day or less never exhibited neurologic signs. The central nervous system (CNS) of affected dogs exhibited endothelial degeneration and hemorrhagic encephalopathy. Focal extravasation of horseradish peroxidase occurred frequently (6/8) in the retrolaminar optic nerve of asymptomatic or clinically affected dogs given 180 mg/kg/day lovastatin, with endothelial degeneration and discrete optic nerve degenerative lesions interpreted as ischemic. The association between the degree of hypocholesterolemia and occurrence of clinical signs was not exact. Total brain cholesterol was similar in treated and control dogs. Hypocholesterolemic dogs had proportionally lowered serum concentrations of alpha-tocopherol, but oral supplementation of this vitamin did not prevent the neurologic syndrome. Endothelial degeneration in the CNS and optic nerve may have reflected in vitro morphologic effects of HMG CoA reductase inhibitors due to extreme inhibition of nonsterol isoprene synthesis. Retinogeniculate axonal (Wallerianlike) degeneration occurred in ≥12% of dogs given 60 mg/kg/day or more lovastatin, with central chromatolysis of occasional retinal ganglion cells. These neuroaxonal changes may have been secondary to vascular effects, but superimposed direct neurotoxic action at the high dosage levels of lovastatin could not be excluded. There was no

  10. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions.

    PubMed Central

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-01-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  11. On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme.

    PubMed

    Leopoldini, Monica; Malaj, Naim; Toscano, Marirosa; Sindona, Giovanni; Russo, Nino

    2010-10-13

    Density functional theory was applied to study the binding mode of new flavonoids as possible inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), an enzyme that catalyzes the four-electron reduction of HMGCoA to mevalonate, the committed step in the biosynthesis of sterols. The investigated flavonoid conjugates brutieridin and melitidin were recently quantified in the bergamot fruit extracts and identified to be structural analogues of statins, lipids concentration lowering drugs that inhibit HMGR. Computations allowed us to perform a detailed analysis of the geometrical and electronic features affecting the binding of these compounds, as well as that of the excellent simvastatin drug, to the active site of the enzyme and to give better insight into the inhibition process.

  12. Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes.

    PubMed

    Yamamoto, A; Hoshi, K; Ichihara, K

    1998-11-13

    We investigated the effect of fluvastatin sodium (fluvastatin) and pravastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, on the formation of thiobarbituric acid reactive substances both in vivo and in vitro in rat liver microsomes and on active oxygen species. Oral administration of fluvastatin at low doses (3.13 and 6.25 mg/kg) inhibited the formation of thiobarbituric acid reactive substances in rat liver microsomes, but high doses (12.5 and 25 mg/kg) did not change the formation of thiobarbituric acid reactive substances. Fluvastatin at any dose used had no effect on the content of cytochrome P-450 and the activity of NADPH-cytochrome P-450 reductase. In in vitro experiments, concentrations of fluvastatin ranging from 1 x 10(-6) - 1 x 10(-4) M markedly inhibited NADPH-dependent lipid peroxidation in liver microsomes, but pravastatin weakly inhibited lipid peroxidation. The order of magnitude of inhibition of each drug on in vitro lipid peroxidation was butylated hydroxytoluene > probucol > or = fluvastatin > pravastatin. Moreover, fluvastatin chemically scavenged active oxygen species such as hydroxyl radicals and superoxide anion generated by the Fenton reaction and by the xanthine-xanthine oxidase system, respectively, but pravastatin showed no scavenging of superoxide anion. These results indicate that the suppression of in vivo and in vitro lipid peroxidation in liver microsomes may be, at least in part, due to the scavenging by fluvastatin of free radicals.

  13. Contribution of Accelerated Degradation to Feedback Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Cholesterol Metabolism in the Liver.

    PubMed

    Hwang, Seonghwan; Hartman, Isamu Z; Calhoun, Leona N; Garland, Kristina; Young, Gennipher A; Mitsche, Matthew A; McDonald, Jeffrey; Xu, Fang; Engelking, Luke; DeBose-Boyd, Russell A

    2016-06-24

    Accumulation of sterols in endoplasmic reticulum membranes stimulates the ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which catalyzes a rate-limiting step in synthesis of cholesterol. This ubiquitination marks HMGCR for proteasome-mediated degradation and constitutes one of several mechanisms for feedback control of cholesterol synthesis. Mechanisms for sterol-accelerated ubiquitination and degradation of HMGCR have been elucidated through the study of cultured mammalian cells. However, the extent to which these reactions modulate HMGCR and contribute to control of cholesterol metabolism in whole animals is unknown. Here, we examine transgenic mice expressing in the liver the membrane domain of HMGCR (HMGCR (TM1-8)), a region necessary and sufficient for sterol-accelerated degradation, and knock-in mice in which endogenous HMGCR harbors mutations that prevent sterol-induced ubiquitination. Characterization of transgenic mice revealed that HMGCR (TM1-8) is appropriately regulated in the liver of mice fed a high cholesterol diet or chow diet supplemented with the HMGCR inhibitor lovastatin. Ubiquitination-resistant HMGCR protein accumulates in the liver and other tissues disproportionately to its mRNA, indicating that sterol-accelerated degradation significantly contributes to feedback regulation of HMGCR in vivo Results of these studies demonstrate that HMGCR is subjected to sterol-accelerated degradation in the liver through mechanisms similar to those established in cultured cells. Moreover, these studies designate sterol-accelerated degradation of HMGCR as a potential therapeutic target for prevention of atherosclerosis and associated cardiovascular disease.

  14. Identification of new flavonol O-glycosides from indigo (Polygonum tinctorium Lour) leaves and their inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed

    Kimura, Hideto; Tokuyama, Shota; Ishihara, Tomoe; Ogawa, Satoshi; Yokota, Kazushige

    2015-04-10

    Indigo plant (Polygonum tinctorium Lour) has been utilized as a medicinal plant with a variety of biological activities. We have recently detected higher levels of flavonoids in indigo leaves. This study was undertaken to conduct the simultaneous analysis of those flavonoids using total extracts from indigo leaves by ultra-performance liquid chromatography-electrospray ionization-time-of-flight/mass spectrometry(E) (UPLC-ESI-TOF/MS(E)). The analysis by UPLC-ESI-TOF/MS(E) allowed us to determine 11 peaks of flavonoid species. The chemical structures of these compounds were identified as flavonol O-glycosides with different types of aglycones by the combination of spectroscopic and chemical methods. The predominant compounds were flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone as an aglycone. Of these, three compounds were elucidated as new compounds. All the isolated flavonol O-glycosides exhibited the inhibitory activity against 3-hydroxy-3-methylglutaryl-CoA reductase in a dose-dependent manner with different potencies. Taken together, our results suggest the potential usefulness of the major flavonol O-glycosides from indigo leaves in controlling cholesterol biosynthesis.

  15. Fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, suppresses very low-density lipoprotein secretion in puromycin aminonucleoside-nephrotic rats.

    PubMed

    Moritomo, Y; Hirano, T; Ebara, T; Kurokawa, M; Naito, H; Furukawa, S; Nagano, S

    1994-01-01

    The effects of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the hyperlipidemia associated with nephrosis were studied. Nephrotic rats, induced by a single intraperitoneal injection of puromycin aminonucleoside (100 mg/kg body weight), had significantly higher plasma triglyceride (TG), total cholesterol and apoprotein (apo) B concentrations than controls. Fluvastatin was administrated as a 0.01% solution in drinking water for 14 days to either normal control or nephrotic rats. Concentrations of TG and apo B in plasma, and very low-density lipoprotein (VLDL) in nephrosis were completely normalized by the treatment with fluvastatin, but concentrations of cholesterol in plasma and each lipoprotein fraction were not altered by the treatment. The ratio of apo E to C in VLDL was significantly decreased in nephrotic rats, but the fluvastatin treatment increased this ratio. TG secretion rate estimated by the Triton WR1339 method was significantly increased in nephrotic rats, but was normalized by fluvastatin. Percent composition of TG in newly secreted VLDL particles in post-Triton plasma was not decreased by fluvastatin treatment, suggesting that the number of newly secreted VLDL particles was reduced by the treatment. Postheparin plasma lipolytic activities were not affected by the fluvastatin treatment. These results demonstrate that fluvastatin can effectively ameliorate the high concentration of VLDL by suppressing the hepatic secretion in nephrotic rats, and suggest that an inhibition of cholesterol biosynthesis suppresses VLDL secretion from the liver.

  16. Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

    PubMed Central

    Xu, Jun-Wei; Xu, Yi-Ning

    2012-01-01

    Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway. PMID:22941092

  17. Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    van Deenen, Nicole; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Schulze Gronover, Christian

    2012-04-01

    Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

  18. Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation

    SciTech Connect

    Rodriguez-Concepcion, M.; Gruissem, W.

    1999-01-01

    Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, the authors manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Their results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

  19. (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level.

    PubMed Central

    Wang, Y L; Beach, M J; Rodwell, V W

    1989-01-01

    We have cloned and sequenced a 505-base-pair (bp) segment of DNA situated upstream of mvaA, the structural gene for (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88) of Pseudomonas mevalonii. The DNA segment that we characterized includes the promoter region for the mva operon. Nuclease S1 mapping and primer extension analysis showed that mvaA is the promoter-proximal gene of the mva operon. Transcription initiates at -56 bp relative to the first A (+1) of the translation start site. Transcription in vivo was induced by mevalonate. Structural features of the mva promoter region include an 80-bp A + T-rich region, and -12, -24 consensus sequences that resemble sequences of sigma 54 promoters in enteric organisms. The relative amplitudes of catalytic activity, enzyme protein, and mvaA mRNA are consistent with a model of regulation of this operon at the transcriptional level. Images PMID:2477360

  20. The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: a proposal as a therapeutic target and as a study model.

    PubMed

    Andrade-Pavón, Dulce; Sánchez-Sandoval, Eugenia; Rosales-Acosta, Blanca; Ibarra, José Antonio; Tamariz, Joaquín; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

    2014-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  1. Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway).

    PubMed

    Wang, Yechun; Guo, Binhui; Zhang, Fei; Yao, Hongyan; Miao, Zhiqi; Tang, Kexuan

    2007-11-30

    The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.

  2. Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat.

    PubMed Central

    Smith, R A; Middleton, B; West, D W

    1986-01-01

    'Expressed' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. 'Expressed' activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. 'Total' activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both 'expressed' and 'total' activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the 'expressed' activity that was close to the nadir value. 'Expressed' activity was lower than 'total' at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the 'expressed'/'total' activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in 'expressed' HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low 'total' HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation. PMID:3814075

  3. Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum

    PubMed Central

    1992-01-01

    We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer- Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum. PMID:1374417

  4. Human carotid atherosclerotic lesion protein components decrease cholesterol biosynthesis rate in macrophages through 3-hydroxy-3-methylglutaryl-CoA reductase regulation.

    PubMed

    Cohen, Elad; Aviram, Michael; Khatib, Soliman; Rosenblat, Mira; Vaya, Jacob

    2015-01-01

    Atherosclerosis is characterized by the formation of cholesterol-loaded macrophages, which are turned into foam cells, the hallmark of early atherogenesis. As part of ongoing research on the interactions among human carotid lesion components and blood elements, the effect of plaque homogenate on macrophage cholesterol biosynthesis rate was examined. Human carotid plaques were ground, extracted with phosphate-buffered saline (homogenate), and then added to the macrophage medium. This extract decreased macrophage cholesterol biosynthesis rate up to 50% in a dose-dependent manner. Cholesterol or lipoproteins were separated from the homogenate and added to the MQ medium. Unlike the homogenate, neither free cholesterol nor the lipoproteins were able to inhibit cholesterol biosynthesis rate under the above experimental concentration, suggesting that the homogenate-induced cholesterol biosynthesis inhibition in our experimental system was not owing to the feedback inhibition of cholesterol. Furthermore, the homogenate remaining after lipoprotein removal (lipoprotein-deficient homogenate) also decreased cholesterol biosynthesis rate, whereas boiled homogenate or phospholipids extracted from the homogenate decreased macrophage cholesterol biosynthesis rate only partially. Finally, cholesterol biosynthesis inhibition was achieved only upon using the precursor [(3)H]acetate, but not [(14)C]mevalonate, suggesting that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA Reductase), the rate-limiting enzyme in the cholesterol biosynthesis pathway, is involved in the above antiatherogenic effect of the homogenate, whereas the treatment with homogenate decreased HMGCoA Reductase mRNA. Proteins and phospholipids from human carotid lesion homogenate decrease cholesterol biosynthesis rate in macrophages secondary to HMGCoA Reductase feedback regulation. Such an effect may delay foam cell formation and atherosclerosis progression.

  5. Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase expression by Zingiber officinale in the liver of high-fat diet-fed rats.

    PubMed

    Nammi, Srinivas; Kim, Moon S; Gavande, Navnath S; Li, George Q; Roufogalis, Basil D

    2010-05-01

    Zingiber officinale has been used to control lipid disorders and reported to possess remarkable cholesterol-lowering activity in experimental hyperlipidaemia. In the present study, the effect of a characterized and standardized extract of Zingiber officinale on the hepatic lipid levels as well as on the hepatic mRNA and protein expression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated in a high-fat diet-fed rat model. Rats were treated with an ethanol extract of Zingiber officinale (400 mg/kg) extract along with a high-fat diet for 6 weeks. The extract of Zingiber officinale significantly decreased hepatic triglyceride and tended to decrease hepatic cholesterol levels when administered over 6 weeks to the rats fed a high-fat diet. We found that in parallel, the extract up-regulated both LDL receptor mRNA and protein level and down-regulated HMG-CoA reductase protein expression in the liver of these rats. The metabolic control of body lipid homeostasis is in part due to enhanced cholesterol biosynthesis and reduced expression of LDL receptor sites following long-term consumption of high-fat diets. The present results show restoration of transcriptional and post-transcriptional changes in low-density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a high-fat diet and provide a rational explanation for the effect of ginger in the treatment of hyperlipidaemia.

  6. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    SciTech Connect

    Ness, Gene C.; Edelman, Jeffrey L.; Brooks, Patricia A.

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  7. Differential effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on the development of myopathy in young rats.

    PubMed

    Reijneveld, J C; Koot, R W; Bredman, J J; Joles, J A; Bär, P R

    1996-06-01

    HMG-CoA reductase inhibitors (statins), cholesterol-lowering drugs that have not been approved for use in children and adolescents, may cause myopathy as a side effect. We compared the effects of three statins (simva-, prava- and lovastatin) in young rats to determine whether skeletal muscle of young animals is more susceptible than that of adults. We also evaluated whether the type of statin (lipophilic versus hydrophilic) determines the degree of muscle damage. Administration via chow of simvastatin (15 mg/kg of body weight/d) and lovastatin (43-55 mg/kg of body weight/d), both lipophilic, caused stunted growth, high creatine kinase (CK) activity in plasma, and severe myopathy. Statin doses that caused damage were much lower for young rats than for adults. Pravastatin (8-55 mg/kg of body weight/d), a hydrophilic drug, caused none of these symptoms. Histologic analysis of hind paw muscles of simvastatin-and lovastatin-treated rats showed abundant signs of damage (hypercontraction, fiber necrosis) in the extensor digitorum longus, correlating with the symptoms noted above. No cellular infiltrates were seen at the onset, pointing to a noninflammatory myopathy. Pravastatin-treated rats never showed signs of myopathy. Impaired DNA synthesis may explain why muscle toxicity is seen at lower doses in young, rapidly developing rats than in adult animals. The differences in muscle damage between the statins may be attributed to differences in lipophilicity and thus in tissue selectivity. Our results can be important when considering drug therapy in young patients with inherited lipoprotein disorders.

  8. Synthesis, biological profile, and quantitative structure-activity relationship of a series of novel 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

    PubMed

    Sit, S Y; Parker, R A; Motoc, I; Han, W; Balasubramanian, N; Catt, J D; Brown, P J; Harte, W E; Thompson, M D; Wright, J J

    1990-11-01

    A series of 9,9-bis(4-fluorophenyl)-3,5-dihydroxy-8-(alkyltetrazol-5-yl)- 6,8-nonadienoic acid derivatives 1 were synthesized and found to inhibit competitively the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The analogues having 1N-methyltetrazol-5-yl attached to the C8-position (3a, 4a, R1 = R2 = F) are the most active in suppressing cholesterol biosynthesis in both in vitro and in vivo models: the IC50 for the chiral form of 3a is 19 nM, Ki = 4.3 x 10(-9)M when Km for HMG-CoA is 28 x 10(-6) M;1 the ED50 (oral) value corresponding to the lactone derivative (4a, BMY 22089) is approximately 0.1 mg/kg. Further, BMY 21950 is nearly 2 orders of magnitude more active in parenchymal heptaocytes, from which most of the serum cholesterol originates, than in other cell preparations (such as spleen, testes, ileum, adrenal, and ocular lens epithelial cells; Table III). This apparent tissue specificity may be highly beneficial since the blocking of cholesterol biosynthesis in other vital organs could eventually lead to undesirable side effects. In addition to the chemical synthesis and biological evaluation, a theoretical study aimed at relating the HMG-CoA reductase inhibitory potency to the three-dimensional structure of the inhibitors was undertaken. With a combination of molecular mapping and 3D-QSAR techniques, it was possible to determine a logical candidate for the conformation of the bound inhibitor and to quantitatively relate inhibitory potency to the shape and size of both the binding site and the C8-substituent.

  9. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    SciTech Connect

    Wang, S.P.; Robert, M.F.; Mitchell, G.A.

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  10. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and application of statins as a novel effective therapeutic approach against Acanthamoeba infections.

    PubMed

    Martín-Navarro, Carmen María; Lorenzo-Morales, Jacob; Machin, Rubén P; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K; Valladares, Basilio; Piñero, José E

    2013-01-01

    Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease.

  11. Ketanserin, an antidepressant, exerts its antileishmanial action via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme of Leishmania donovani.

    PubMed

    Singh, Sushma; Dinesh, Neeradi; Kaur, Preet Kamal; Shamiulla, Baigadda

    2014-06-01

    Leishmaniasis is one of the major health problems existing globally. The current chemotherapy for leishmaniasis presents several drawbacks like toxicity and increased resistance to existing drugs, and hence, there is a necessity to look out for the novel drug targets and new chemical entities. Current trend in drug discovery arena is the "repurposing" of old drugs for the treatment of diseases. In the present study, an antidepressant, ketanserin, was found lethal to both Leishmania donovani promastigotes and intracellular amastigotes with no apparent toxicity to the cells. Ketanserin killed promastigotes and amastigotes with an IC50 value of 37 μM and 28 μM respectively, in a dose-dependent manner. Ketanserin was found to inhibit L. donovani recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme with an IC50 value of 43 μM. Ketanserin treated promastigotes were exogenously supplemented with sterols like ergosterol and cholesterol to rescue cell death. Ergosterol could recover the inhibition partially, whereas cholesterol supplementation completely failed to rescue the inhibited parasites. Further, HMGR-overexpressing parasites were generated by transfecting Leishmania promastigotes with an episomal pspα hygroα-HMGR construct. Wild-type and HMGR overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. The HMGR overexpressors showed twofold resistance to ketanserin. These observations suggest that the lethal effect of ketanserin is due to inhibition of HMGR, the rate-limiting enzyme of the ergosterol biosynthetic pathway. Since targeting of the sterol biosynthetic pathway enzymes may be useful therapeutically, the present study may have implications in treatment of leishmaniasis.

  12. Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells1[OPEN

    PubMed Central

    Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles

    2015-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells. PMID:26015445

  13. Immune-mediated myopathy related to anti 3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies as an emerging cause of necrotizing myopathy induced by statins.

    PubMed

    Lahaye, Clément; Beaufrére, Anne Marie; Boyer, Olivier; Drouot, Laurent; Soubrier, Martin; Tournadre, Anne

    2014-01-01

    Immune-mediated necrotizing myopathy (IMNM) associated with statin use and anti 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibody is a new and emerging entity that supports a link between statin use and IMNM and raises the questions of distinct clinical phenotypes and treatment strategy. We describe the clinical and histopathological characteristics of a patient and discuss the spectrum of IMNM and statin-induced myopathies. A 65-year-old man was suffering from proximal muscle weakness and elevated CK levels, following exposure to statin therapy. The symptoms worsened despite discontinuation of the drug. At that point, no myositis-specific or -associated antibodies were detected. Malignancy screening did not reveal abnormalities. Muscle biopsy demonstrated a predominantly necrotizing myopathy with minimal lymphocytic infiltrates, MHC class I expression in necrotic muscle fibers, and complement deposition on scattered non-necrotic muscle fibers. Muscle protein analysis by western blot was normal. The patient did not improve with steroid and methotrexate and required monthly intravenous immunoglobulin (IVIG) therapy. Muscle strength gradually improved, CK levels normalized and IVIG were stopped 1 year later. Screening for anti-HMGCR antibodies, not available at the time of presentation, was highly positive. Identification of anti-HMGCR antibodies in statin-exposed patients with myopathy appears to be helpful both for differential diagnosis and for treatment strategy. In patients who did not improve after discontinuation of the statin treatment, a muscle biopsy should be performed as well as screening for anti-HMGCR antibodies. Patients with this disorder require aggressive immunosuppressive treatment.

  14. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid.

    PubMed Central

    Choi, D; Ward, B L; Bostock, R M

    1992-01-01

    Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species. PMID

  15. Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.

    PubMed

    Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Leivar, Pablo; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles; Campos, Narciso

    2015-07-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells.

  16. Statins and myotoxic effects associated with anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies: an observational study in Japan.

    PubMed

    Watanabe, Yurika; Suzuki, Shigeaki; Nishimura, Hiroaki; Murata, Ken-ya; Kurashige, Takashi; Ikawa, Masamichi; Asahi, Masaru; Konishi, Hirofumi; Mitsuma, Satsuki; Kawabata, Satoshi; Suzuki, Norihiro; Nishino, Ichizo

    2015-01-01

    Statins have a variety of myotoxic effects and can trigger the development of inflammatory myopathies or myasthenia gravis (MG) mediated by immunomodulatory properties. Autoantibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have been identified in patients with statin-associated myopathy. The purpose of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) of anti-HMGCR antibodies and to elucidate the clinical significance of anti-HMGCR antibodies in Japanese patients with inflammatory myopathies or MG. We enrolled 75 patients with inflammatory myopathies, who were all negative for anti-signal recognition particle and anti-aminoacyl transfer RNA synthetase antibodies. They were referred to Keio University and National Center of Neurology and Psychiatry between October 2010 and September 2012. We also studied 251 patients with MG who were followed at the MG Clinic at Keio University Hospital. Anti-HMGCR antibodies were detected by ELISA. We investigated demographic, clinical, radiological, and histological findings associated with anti-HMGCR antibodies. We established the anti-HMGCR ELISA with the recombinant protein. Protein immunoprecipitation detected autoantigens corresponding to HMGCR. Immunohistochemistry using muscle biopsy specimens revealed regenerating muscle fibers clearly stained by polyclonal anti-HMGCR antibodies and patients' serum. Anti-HMGCR autoantibodies were specifically detected in 8 patients with necrotizing myopathy. The seropositivity rate in the necrotizing myopathy patients was significantly higher than those in the patients with other histological diagnoses of inflammatory myopathies (31% vs 2%, P = 0.001). Statins were administered in only 3 of the 8 anti-HMGCR-positive patients. Myopathy associated with anti-HMGCR antibodies showed mild limb weakness and favorable response to immunotherapy. All 8 patients exhibited increased signal intensities on short T1 inversion recovery of muscle MRI. Of

  17. Cholesterol-lowering effect of NK-104, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, in guinea pig model of hyperlipidemia.

    PubMed

    Aoki, T; Yamazaki, H; Suzuki, H; Tamaki, T; Sato, F; Kitahara, M; Saito, Y

    2001-01-01

    Although benefits of statins have been demonstrated even in normolipidemic patients at high risk, the main target of statin therapy is the hypercholesterolemic patient. The aim of this study was to examine the hypocholesterolemic effect of NK-104 ((+)-monocalcium bis((3R,5S,6S)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]- 3,5-dihydroxy-6-heptenoate), CAS 147526-32-7), a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and its mechanism of action in hypercholesterolemic animals. In guinea pigs fed a diet containing 15% (w/w) fat rich in laurate for 6 weeks, the liver cholesterol content was markedly increased and plasma total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and LDL-apoB were elevated 4.8, 5.2 and 1.7 times, respectively, compared with normal diet fed animals. These changes were maintained by reduced LDL clearance in the presence of markedly cholesterol-enriched LDL in the plasma. In this model, the LDL-C reduction rates by 0.1, 0.3 and 1 mg/kg of NK-104 orally administered for 2 weeks (from week 4 to week 6), were 11, 27 and 32%, respectively, from controls, being similar in normal guinea pigs previously examined. Those for 3 and 10 mg/kg of atorvastatin (CAS 134523-00-5) were 25 and 39%, respectively. Thus about 10 times higher doses of atorvastatin were required than of NK-104 to cause a similar cholesterol-lowering effect. This reduction of plasma cholesterol was accompanied by an improvement of LDL clearance (24 and 47% increase in fractional catabolic rate by 1 mg/kg of NK-104 and 10 mg/kg of atorvastatin, respectively) and LDL composition. In conclusion, in guinea pig hypercholesterolemia caused by high-laurate diet, NK-104 and atorvastatin lowered plasma cholesterol levels with an improvement of LDL composition and with an increase in LDL clearance, presumably through reduction of the liver cholesterol content, although hepatic cholesterol synthesis might have been markedly suppressed in this model.

  18. Enhancement of sterol synthesis by the monoterpene perillyl alcohol is unaffected by competitive 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

    PubMed

    Cerda, S R; Wilkinson, J; Branch, S K; Broitman, S A

    1999-06-01

    Monoterpenes such as limonene and perillyl alcohol (PA) are currently under investigation for their chemotherapeutic properties which have been tied to their ability to affect protein isoprenylation. Because PA affects the synthesis of isoprenoids, such as ubiquinone, and cholesterol is the end product of the synthetic pathway from which this isoprenoid pathway branches, we investigated the effects of this compound upon cholesterol metabolism in the colonic adenocarcinoma cell line SW480. PA (1 mM) inhibited incorporation of 14C-mevalonate into 21-26 kDa proteins by 25% in SW480 cells. Cholesterol (CH) biosynthesis was assessed by measuring the incorporation of 14C-acetate and 14C-mevalonate into 27-carbon-sterols. Cells treated with PA (1 mM) exhibited a fourfold increase in the incorporation of 14C-acetate but not 14C-mevalonate into cholesterol. Mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, at 2 microM concentration, inhibited CH synthesis from 14C-acetate by 80%. Surprisingly, concurrent addition of mevinolin and PA did not significantly alter the stimulatory effects of PA. As observed differences in 14C-acetate and 14C-mevalonate precursor labeling could indicate PA affects early pathway events, the effects of this monoterpene on HMG-CoA reductase activity were evaluated. Unexpectedly, 1 mM PA did not stimulate activity of this enzyme. Consistent with its action as a reversibly bound inhibitor, in washed microsomes, 2 microM mevinolin pretreatment increased reductase protein expression causing a 12.7 (+/- 2.4)-fold compensatory HMG-CoA reductase activity increase; concurrent treatment with 1 mM PA attenuated this to a 5.3 (+/- 0.03)-fold increase. Gas chromatographic analysis confirmed CH was the major lipid present in the measured thin-layer chromatography spot. Since 14C-acetate incorporation into free fatty acid and phospholipid pools was not significantly affected by PA treatment, nonspecific changes in whole

  19. Inhibition of cholesterol synthesis ex vivo and in vivo by fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

    PubMed

    Yamamoto, A; Itoh, S; Hoshi, K; Ichihara, K

    1995-03-15

    The inhibitory effect of fluvastatin sodium (fluvastatin), a new type of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A inhibitor, on de novo cholesterol synthesis was investigated and compared with that of pravastatin. Fluvastatin at a concentration of 12.5 mg/kg inhibited sterol synthesis ex vivo from [14C]acetate in rat liver and ileum by 97-99% with respect to the control, while the inhibition in kidney was 55%. The inhibition by fluvastatin in the liver and ileum persisted for approximately 9 h after administration. Significant differences between fluvastatin also had an inhibitory effect on cholesterol synthesis in vivo in various tissues of rats given [14C]acetate intraperitoneally. Sterol synthesis in the liver, ileum and kidney was inhibited by over 95% 3 h after administration of 6.25 mg/kg of fluvastatin. Significant differences between fluvastatin and pravastatin were found in the liver and ileum. Fluvastatin was more potent than pravastatin in inhibiting both ex vivo and in vivo sterol synthesis in the ileum (but not in kidney) and liver.

  20. Novel synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity that inhibit tumor cell proliferation and are structurally unrelated to existing statins.

    PubMed

    Perchellet, Jean-Pierre H; Perchellet, Elisabeth M; Crow, Kyle R; Buszek, Keith R; Brown, Neil; Ellappan, Sampathkumar; Gao, Ge; Luo, Diheng; Minatoya, Machiko; Lushington, Gerald H

    2009-11-01

    Pilot-scale libraries of eight-membered medium ring lactams (MRLs) and related tricyclic compounds (either seven-membered lactams, thiolactams or amines) were screened for their ability to inhibit the catalytic activity of human recombinant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in vitro. A dozen of the synthetic compounds mimic the inhibition of purified HMG-CoA reductase activity caused by pravastatin, fluvastatin and sodium salts of lovastatin, mevastatin and simvastatin in this cell-free assay, suggesting direct interaction with the rate-limiting enzyme of cholesterol biosynthesis. Moreover, several MRLs inhibit the metabolic activity of L1210 tumor cells in vitro to a greater degree than fluvastatin, lovastatin, mevastatin and simvastatin, whereas pravastatin is inactive. Although the correlation between the concentration-dependent inhibitions of HMG-CoA reductase activity over 10 min in the cell-free assay and L1210 tumor cell proliferation over 4 days in culture is unclear, some bioactive MRLs elicit interesting combinations of statin-like (IC50: 7.4-8.0 microM) and anti-tumor (IC50: 1.4-2.3 microM) activities. The HMG-CoA reductase-inhibiting activities of pravastatin and an MRL persist in the presence of increasing concentrations of NADPH. But increasing concentrations of HMG-CoA block the HMG-CoA reductase-inhibiting activity of pravastatin without altering that of an MRL, suggesting that MRLs and existing statins may have different mechanisms of enzyme interaction and inhibition. When tested together, suboptimal concentrations of synthetic MRLs and existing statins have additive inhibitory effects on HMG-CoA reductase activity. Preliminary molecular docking studies with MRL-based inhibitors indicate that these ligands fit sterically well into the HMG-CoA reductase statin-binding receptor model and, in contrast to mevastatin, may occupy a narrow channel housing the pyridinium moiety on NADP+.

  1. Involvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.).

    PubMed Central

    Choi, D.; Bostock, R. M.

    1994-01-01

    A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested. PMID:12232162

  2. Modulation of hepatic apolipoprotein B, 3-hydroxy-3-methylglutaryl-CoA reductase and low-density lipoprotein receptor mRNA and plasma lipoprotein concentrations by defined dietary fats. Comparison of trimyristin, tripalmitin, tristearin and triolein.

    PubMed Central

    Bennett, A J; Billett, M A; Salter, A M; Mangiapane, E H; Bruce, J S; Anderton, K L; Marenah, C B; Lawson, N; White, D A

    1995-01-01

    Different dietary fatty acids exert specific effects on plasma lipids but the mechanism by which this occurs is unknown. Hamsters were fed on low-cholesterol diets containing triacylglycerols enriched in specific saturated fatty acids, and effects on plasma lipids and the expression of genes involved in hepatic lipoprotein metabolism were measured. Trimyristin and tripalmitin caused significant rises in low-density lipoprotein (LDL) cholesterol which were accompanied by significant reductions in hepatic LDL receptor mRNA levels. Tripalmitin also increased hepatic expression of the apolipoprotein B gene, implying an increased production of LDL via very-low-density lipoprotein (VLDL) and decreased removal of LDL in animals fed this fat. Hepatic levels of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not vary significantly between the groups. Compared with triolein, tristearin had little effect on hepatic gene expression or total plasma cholesterol. However, it caused a marked decrease in VLDL cholesterol and a rise in LDL cholesterol such that overall it appeared to be neutral. Lipid analysis suggested a rapid desaturation of much of the dietary stearate. The differential changes in plasma lipids and hepatic mRNA levels induced by specific dietary fats suggests a role for fatty acids or a metabolite thereof in the regulation of the expression of genes involved in lipoprotein metabolism. PMID:7575449

  3. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

    PubMed Central

    Hernández-Perera, O; Pérez-Sala, D; Navarro-Antolín, J; Sánchez-Pascuala, R; Hernández, G; Díaz, C; Lamas, S

    1998-01-01

    Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors. PMID:9637705

  4. Inhibition of hepatic S-3-hydroxy-3-methylglutaryl-CoA reductase and in vivo rates of lipogenesis by a mixture of pure cyclic monoterpenes.

    PubMed

    Middleton, B; Hui, K P

    1982-09-15

    A proprietary mixture of pure cyclic monoterpenes (Rowachol) inhibited hepatic HMGCoA reductase by 50-60% when measured 17 hr after the oral administration of a single dose to rats. The extent of this inhibition was independent of the normal activity range of HMGCoA reductase within its diurnal cycle and the same inhibition (65%) was found in 24 hr starved animals where the control reductase activity was less than 20% that of normal fed rats. De novo sterol and fatty acid synthesis in intact, fed rats was measured by incorporation of 3H from injected H2O. In rats treated with Rowachol the rate of sterol synthesis in vivo was inhibited 52% in liver and 44% in testis with no significant effects in other tissues. The synthesis of non sterol (isoprenoid) compounds in testis was unaffected and the inhibition of sterol synthesis in this tissue probably reflects decreased acquisition of newly synthesized material from liver rather than any effect on the endogenous process. In the same animals the rate of fatty acid synthesis was inhibited 55% in liver. These effects were associated with a significant depletion of liver glycogen which may account for the reduction in rate of fatty acid synthesis. We conclude that the reported cholelitholytic action of monoterpenes is associated with the physiological inhibition of hepatic sterol synthesis mediated by decrease HMGCoA reductase activity.

  5. Application of modified Lineweaver-Burk plots to studies of kinetics and regulation of radish 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed

    Bach, T J; Lichtenthaler, H K

    1984-06-06

    We propose the use of modified Lineweaver-Burk plots for the correct evaluation of Michaelis-Menten parameters in radioactive enzyme assays. A correction factor X for the translation of 1/S0 into 1/S is directly derived from the integrated Michaelis-Menten equation without the need of complicated calculations. In practice, this approach is favorably combined with an isotope dilution method which enhances the reliability of measurements at low substrate concentrations. The usefulness of the theoretical and practical approach is demonstrated in investigations of HMG-CoA reductase present in membrane fractions isolated from radish seedlings. The enzyme in the two main membrane fractions obtained by centrifugation at 16000 X g ( P16000 ) and at 105000 X g ( P105000 ) appears to be independently regulated by phytochrome and by phytohormones. Whereas active phytochrome decreases the apparent V of HMG-CoA reductase in the P105000 without affecting the Km, it increases the apparent Km in the P16000 . Kinetin treatment also results in a higher apparent Km of the enzyme in the P16000 fraction. Gibberellic acid and indoleacetic acid did not exhibit such a clear effect.

  6. Beneficial effects of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, on cardiac function in ischemic and nonischemic heart failure.

    PubMed

    Aoyagi, Teruhiko; Nakamura, Fumitaka; Tomaru, Takanobu; Toyo-Oka, Teruhiko

    2008-01-01

    HMG-CoA reductase inhibitors (statins) have recently been reported to improve cardiac function, and decrease the incidence of heart failure (HF) in hyperlipidemic patients. However, evidence for statin treatment in patients with HF remains a subject of debate. Thus, a study was initiated to examine the effects of pitavastatin on cardiac function evaluated by echocardiographic findings and plasma brain natriuretic peptide (BNP) levels in patients with HF. Twenty-three patients with HF were treated with pitavastatin 1-2 mg/day in addition to standard therapy for 7.5 +/- 3.8 months. Left ventricular end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) were determined by echocardiography. Left ventricular ejection fraction (LVEF) was calculated using Teichholz's formula. Serum lipid and plasma BNP levels were also measured. During the follow-up period, LVEF was increased from 42 +/- 11 to 48 +/- 13% (P = 0.002). LVDs was reduced from 43 +/- 10 to 40 +/- 10 mm (P < 0.001), while there was no change in LVDd. E/A (n = 10) and deceleration time (n = 7), obtained in some patients, did not change significantly (0.89 +/- 0.33 to 0.77 +/- 0.17%, and 215 +/- 46 to 227 +/- 72 msec, respectively). In addition, the plasma BNP level was moderately, but significantly decreased from 94 +/- 78 to 70 +/- 56 pg/mL (P = 0.005). In subgroup analysis, LVEF was improved in both patients with ischemic and nonischemic HF. There was no significant correlation between the percent change in serum total cholesterol and the percent change in LVEF by pitavastatin treatment. Serum total cholesterol, LDL-cholesterol, and triglycerides decreased by 21%, 30%, and 15%, respectively, and HDL-cholesterol increased by 12%. Pitavastatin improved cardiac function in patients with HF, which generally worsens with time. The results suggest that pitavastatin may be beneficial for treatment of HF.

  7. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston

    PubMed Central

    2010-01-01

    Background Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. Results A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. Conclusion A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher

  8. Ethanol extract of Zhongtian hawthorn lowers serum cholesterol in mice by inhibiting transcription of 3-hydroxy-3-methylglutaryl-CoA reductase via nuclear factor-kappa B signal pathway.

    PubMed

    Hu, Hai-Jie; Luo, Xue-Gang; Dong, Qing-Qing; Mu, Ai; Shi, Guo-Long; Wang, Qiu-Tong; Chen, Xiao-Ying; Zhou, Hao; Zhang, Tong-Cun; Pan, Li-Wen

    2016-03-01

    Hawthorn is a berry-like fruit from the species of Crataegus. In China, it has another more famous name, Shan-Zha, which has been used to improve digestion as a traditional Chinese medicine or food for thousands of years. Moreover, during the last decades, hawthorn has received more attention because of its potential to treat cardiovascular diseases. However, currently, only fruits of C. pinnatifida and C. pinnatifida var. major are included as Shan-Zha in the Chinese Pharmacopoeia. In this study, our results showed that the ethanol extract of Zhongtian hawthorn, a novel grafted cultivar of C. cuneata (wild Shan-Zha), could markedly reduce body weight and levels of serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, and liver cholesterol of hyperlipidemia mice. It could suppress the stimulation effect of high-fat diet on the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and p65, and counteract the downregulation of CYP7A1 and LDLR. In addition, the results of luciferase reporter assay and Western blot showed that the transcriptional activity of HMGCR promoter was inhibited by Zhongtian hawthorn ethanol extract in a dose-dependent manner, while overexpression of p65 could reverse this transcriptional repression effect. These results suggested that Zhongtian hawthorn could provide health benefits by counteracting the high-fat diet-induced hypercholesteolemic and hyperlipidemic effects in vivo, and the mechanism underlying this event was mainly dependent on the suppressive effect of Zhongtian hawthorn ethanol extract on the transcription of HMGCR via nuclear factor-kappa B (NF-κB) signal pathway. Therefore, this novel cultivar of hawthorn cultivar which has much bigger fruits, early bearing, high yield, cold resistance, and drought resistance, might be considered as a good alternative to Shan-Zha and has great value in the food and medicine industry. In addition, to our best knowledge, this is also the first report that the

  9. Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on ubiquinone levels in rat skeletal muscle and heart: relationship to cytotoxicity and inhibitory activity for cholesterol synthesis in human skeletal muscle cells.

    PubMed

    Yamazaki, Hiroyuki; Suzuki, Mahomi; Aoki, Taro; Morikawa, Shigeru; Maejima, Takashi; Sato, Fumiyasu; Sawanobori, Kimio; Kitahara, Masaki; Kodama, Tatsuhiko; Saito, Yasushi

    2006-12-01

    Although statins are prescribed as relatively safe and effective drugs for hypercholesterolemic patients, it has been reported that a significant side effect, myopathy, occurs infrequently during medication. Moreover, because statins decrease cardiac ubiquinone levels, the risk of cardiac dysfunction has been suggested. This study sought to evaluate and compare the cytotoxicity of statins (cerivastatin, pitavastatin, fluvastatin, simvastatin, atorvastatin and pravastatin) in cultured human skeletal muscle cells (HSkMCs) and the effects on ubiquinone levels in statin-treated rat skeletal muscle and heart. Cerivastatin, the most potent inhibitor of HMG-CoA reductase, showed the strongest cytotoxicity (over 10-fold) among the statins examined, while the effects of the others were in a similar range. In rat experiments, neither pitavastatin nor cerivastatin decreased ubiquinone levels in skeletal muscle, but both dose-dependently lowered ubiquinone levels in the heart. As the rates of reduction by pitavastatin (9.6% at 30 mg/kg) and cerivastatin (9.7% at 0.3 mg/kg) were almost equal, it was estimated that cerivastatin reduced ubiquinone levels in the rat heart approximately 100-fold more strongly than pitavastatin, based on the effective doses. We found that cerivastatin showed the most potent cytotoxicity in HSkMCs and strongly lowered ubiquinone levels in the rat heart.

  10. Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase.

    PubMed

    Casals, Núria; Gómez-Puertas, Paulino; Pié, Juan; Mir, Cecilia; Roca, Ramón; Puisac, Beatriz; Aledo, Rosa; Clotet, Josep; Menao, Sebastián; Serra, Dolors; Asins, Guillermina; Till, Jacqueline; Elias-Jones, Alun C; Cresto, Juan C; Chamoles, Nestor A; Abdenur, Jose E; Mayatepek, Ertan; Besley, Guy; Valencia, Alfonso; Hegardt, Fausto G

    2003-08-01

    This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

  11. Effect of polyphenols on 3-hydroxy-3-methylglutaryl-coenzyme A lyase activity in human hepatoma HepG2 cell extracts.

    PubMed

    Nakagawa, Saori; Kojima, Yuko; Sekino, Koichi; Yamato, Susumu

    2013-01-01

    When carbohydrate metabolism is impaired, fatty acid metabolism is activated. Excess acetyl-coenzyme A (CoA) is generated from fatty acids by β-oxidation and is used for the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and subsequently for acetoacetate. High levels of secreted ketone bodies (acetoacetate and 3β-hydroxybutyrate) lower the pH of blood and urine, resulting in ketoacidosis. HMG-CoA lyase in hepatic cells is a rate-limiting enzyme catalyzing the cleavage of HMG-CoA to acetoacetate, and thus inhibition of this enzyme results in reduced acetoacetate production, in other words, impaired ketoacidosis. Inhibition of HMG-CoA lyase activity possibly prevents ketoacidosis and should be the therapeutic target. Polyphenols are common and abundant dietary constituents with beneficial effects on human health. We examined the inhibitory effects of dietary polyphenols on HMG-CoA lyase activity in cellular extracts of human hepatoma HepG2 cells. Of the nine representative dietary polyphenols tested, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and gallic acid (GA) effectively inhibited HMG-CoA lyase activity. Lineweaver-Burk analysis revealed that EGC and EGCG are likely to be mixed-type noncompetitive inhibitors. Pyrogallol with the gallyl structure also inhibited HMG-CoA lyase activity, suggesting that the gallyl moiety of polyphenols is important for the inhibition of HMG-CoA lyase activity.

  12. Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase

    PubMed Central

    Fernandes, Nicolle V.; Yeganehjoo, Hoda; Katuru, Rajasekhar; DeBose-Boyd, Russell A.; Morris, Lindsey L.; Michon, Renee; Yu, Zhi-Ling; Mo, Huanbiao

    2014-01-01

    The rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation of nuclear lamins and Ras and dolichol-mediated glycosylation of growth factor receptors. The diterpene geranylgeraniol downregulates the level of HMG CoA reductase and suppresses the growth of human liver, lung, ovary, pancreas, colon, stomach, and blood tumors. We evaluated the growth-suppressive activity of geranylgeraniol in human prostate carcinoma cells. Geranylgeraniol induced dose-dependent suppression of the viability of human DU145 prostate carcinoma cells (IC50 = 80 ±18 μmol/L, n =5) following 72-h incubations in 96-well plates. Cell cycle was arrested at the G1 phase with a concomitant decrease in cyclin D1 protein. Geranylgeraniol-induced apoptosis was detected by flow cytometric analysis, fluorescence microscopy following acridine orange and ethidium bromide dual staining, and caspase-3 activation. Geranylgeraniol-induced viability suppression was accompanied by concentration-dependent decrease in the level of HMG CoA reductase protein. As a nonsterol molecule that downregulates HMG CoA reductase in the presence of sterols, geranylgeraniol may have potential in the chemoprevention and/or therapy of human prostate cancer. PMID:24006306

  13. Tissue-specific expression and dietary regulation of chimeric mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase/human growth hormone gene in transgenic mice.

    PubMed

    Serra, D; Fillat, C; Matas, R; Bosch, F; Hegardt, F G

    1996-03-29

    We have studied the role of the mitochondrial 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase gene in regulating ketogenesis. The gene exhibits expression in various tissues and it is regulated in a tissue-specific manner. To investigate the underlying mechanisms of this expression, we linked a 1148-base-pair portion of the mitochondrial HMG-CoA synthase promoter to the human growth hormone (hGH) gene and analyzed the expression of the hGH reporter gene in transgenic mice. mRNA levels of hGH were observed in liver, testis, ovary, stomach, colon, cecum, brown adipose tissue, spleen, adrenal glands, and mammary glands from adult mice, and also in liver and stomach, duodenum, jejunum, brown adipose tissue, and heart of suckling mice. There was no expression either in kidney or in any other nonketogenic tissue. The comparison between these data and those of the endogenous mitochondrial HMG-CoA synthase gene suggests that the 1148 base pairs of the promoter contain the elements necessary for expression in liver and testis, but an enhancer is necessary for full expression in intestine of suckling animals and that a silencer prevents expression in stomach, brown adipose tissue, spleen, adrenal glands, and mammary glands in wild type adult mice. In starvation, transgenic mice showed higher expression in liver than did wild type. Both refeeding and insulin injection reduced the expression. Fat diets, composed in each case of different fatty acids, produced similar expression levels, respectively, to those found in wild type animals, suggesting that long-, medium-, and short-chain fatty acids may exert a positive influence on the transcription rate in this 1148-base-pair portion of the promoter. The ketogenic capacity of liver and the blood ketone body levels were equal in transgenic mice and in nontransgenic mice.

  14. Ketogenic mitochondrial 3-hydroxy 3-methylglutaryl-CoA synthase gene expression in intestine and liver of suckling rats.

    PubMed

    Serra, D; Asins, G; Hegardt, F G

    1993-03-01

    The ketogenic mitochondrial 3-hydroxy 3-methylglutaryl-coenzyme A (HMG-CoA) synthase gene is expressed in intestine of suckling rats, its mRNA levels changing with age. Intestine mitochondrial mRNA values reach maximum levels on the 12th postnatal day and then decrease smoothly. Mother's milk may influence the intestine expression, since mRNA levels at birth are very low, increasing after the first lactation. Moreover, rats weaned at either Day 18 or 21 decrease their mRNA levels dramatically and there is no expression in adult rats. Mitochondrial HMG-CoA synthase is also expressed in liver of suckling rats but the developmental pattern of mRNAs is different from that in intestine, showing the highest values at Day 3 of life. mRNA levels in liver are lower than in intestine for most of the suckling period, suggesting the physiological relevance of the intestine for the ketogenic process of the whole body. Liver mRNA levels on weaning and in adult rats are high enough to sustain hepatic ketogenesis.

  15. Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes.

    PubMed Central

    Ayté, J; Gil-Gómez, G; Haro, D; Marrero, P F; Hegardt, F G

    1990-01-01

    We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass. Identify of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase. (ii) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase. (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity. (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme. The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria. These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme. Images PMID:1971108

  16. Effects of Statins on 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition Beyond Low-Density Lipoprotein Cholesterol

    PubMed Central

    Liao, James K.

    2009-01-01

    Statins are potent inhibitors of cholesterol biosynthesis and exert beneficial effects in the primary and secondary prevention of coronary artery disease. However, the overall benefits observed with statins appear to occur much earlier and to be greater than what might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent or “pleiotropic” effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. Many of these pleiotropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of the small guanosine triphosphate–binding proteins Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the pleiotropic effects of statins. PMID:16126020

  17. The ketogenic diet upregulates expression of the gene encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in rat brain.

    PubMed

    Cullingford, Tim E; Eagles, Douglas A; Sato, Hitoshi

    2002-04-01

    The ketogenic diet is a clinically and experimentally effective anti-epileptic treatment whose molecular mechanism(s) of action remain to be elucidated. As a first step in defining its effects on regulation of fatty acid oxidation and ketogenesis at the genetic level, we have administered to rats: (1) a calorie-restricted ketogenic diet (KCR); (2) a calorie-restricted normal diet (NCR); or (3) a normal diet ad libitum (NAL). We have used RNase protection to co-assay diet-induced changes in abundance of the mRNA encoding the critical enzyme of ketogenesis from acetyl-CoA namely mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) in liver and brain, together with mRNAs encoding three other key enzymes of fatty acid oxidation. We demonstrate that NCR-fed rats exhibit a significant 2-fold increase in liver mHS mRNA compared to NAL-fed rats, and that KCR-fed rats exhibit a significant 2-fold increase in both liver and brain mHS mRNA compared to NAL-fed rats. Our results demonstrate, for the first time, the effect of a ketogenic diet on gene expression in brain, and suggest possible anti-epileptic mechanisms for future investigation.

  18. Cloning, Expression Profiling and Functional Analysis of CnHMGS, a Gene Encoding 3-hydroxy-3-Methylglutaryl Coenzyme A Synthase from Chamaemelum nobile.

    PubMed

    Cheng, Shuiyuan; Wang, Xiaohui; Xu, Feng; Chen, Qiangwen; Tao, Tingting; Lei, Jing; Zhang, Weiwei; Liao, Yongling; Chang, Jie; Li, Xingxiang

    2016-03-08

    Roman chamomile (Chamaemelum nobile L.) is renowned for its production of essential oils, which major components are sesquiterpenoids. As the important enzyme in the sesquiterpenoid biosynthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyze the crucial step in the mevalonate pathway in plants. To isolate and identify the functional genes involved in the sesquiterpene biosynthesis of C. nobile L., a HMGS gene designated as CnHMGS (GenBank Accession No. KU529969) was cloned from C. nobile. The cDNA sequence of CnHMGS contained a 1377 bp open reading frame encoding a 458-amino-acid protein. The sequence of the CnHMGS protein was highly homologous to those of HMGS proteins from other plant species. Phylogenetic tree analysis revealed that CnHMGS clustered with the HMGS of Asteraceae in the dicotyledon clade. Further functional complementation of CnHMGS in the mutant yeast strain YSC6274 lacking HMGS activity demonstrated that the cloned CnHMGS cDNA encodes a functional HMGS. Transcript profile analysis indicated that CnHMGS was preferentially expressed in flowers and roots of C. nobile. The expression of CnHMGS could be upregulated by exogenous elicitors, including methyl jasmonate and salicylic acid, suggesting that CnHMGS was elicitor-responsive. The characterization and expression analysis of CnHMGS is helpful to understand the biosynthesis of sesquiterpenoid in C. nobile at the molecular level and also provides molecular wealth for the biotechnological improvement of this important medicinal plant.

  19. Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.

    PubMed

    Adams, S H; Alho, C S; Asins, G; Hegardt, F G; Marrero, P F

    1997-05-15

    The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.

  20. Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes.

    PubMed

    Nagegowda, Dinesh A; Bach, Thomas J; Chye, Mee-Len

    2004-11-01

    3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1). Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity. Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

  1. In vitro and in vivo biotransformation of simvastatin, an inhibitor of HMG CoA reductase.

    PubMed

    Vickers, S; Duncan, C A; Vyas, K P; Kari, P H; Arison, B; Prakash, S R; Ramjit, H G; Pitzenberger, S M; Stokker, G; Duggan, D E

    1990-01-01

    Simvastatin (SV), an analog of lovastatin, is the lactone form of 1', 2', 6', 7', 8', 8a'-hexahydro-3,5-dihydroxy-2', 6'-dimethyl-8' (2", 2"-dimethyl-1"-oxobutoxy)-1'-naphthalene-heptanoic acid (SVA) which lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase. SV but not its corresponding hydroxy acid form SVA underwent microsomal metabolism. Major in vitro metabolites were 6'-OH-SV (I) and 3"-OH-SV (III) formed by allylic and aliphatic hydroxylation, respectively, and 6'-exomethylene-SV (IV) formed by dehydrogenation. In rats, dogs, and humans, biliary excretion is the major route of elimination. Biliary metabolites (as both hydroxy acids and lactones) also included 6'-CH2OH-SV (V) and 6'-COOH-SV (VI) in both of which the 6'-chiral center had been inverted. High levels of esterase in rodent plasma favored the formation of SVA from SV. The formation of 1', 2', 6', 7', 8', 8a'-hexahydro-2', 6'-dimethyl-8'-(2",2"-dimethyl-1-oxobutoxy)-1'-naphthalene-pentano ic acid (VII) only in rodents represented a species difference in the metabolism of SV. It is proposed that VII is formed by beta-oxidation pathways of fatty acid intermediary metabolism. Several metabolites resulting from microsomal oxidation (after subsequent conversion from lactones to hydroxy acids) are effective inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and may contribute to the cholesterol lowering effect of SV. Qualitatively, the metabolism of SV closely resembles that of lovastatin.

  2. The effect of compactin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, on cholesterogenesis and serum cholesterol levels in rats and chicks.

    PubMed

    Fears, R; Richards, D H; Ferres, H

    1980-04-01

    Compactin, [7-(1,2,6,7,8,8a-hexahydro-2-methyl-8-(2-methylbutyrylox)naphthyl)-3-hydroxyheptan-5-olide], a potent competitive inhibitor of the rate-determining step in cholesterol biosynthesis, was used to study the influence of changes in cholesterogenesis on serum cholesterol levels. Up to 3 h after a single oral dose (20 or 50 mg/kg) or after the last of a series of daily oral doses (50 mg/kg for 7 or 28 days) to young, male normolipidaemic rats, compactin consistently inhibited cholesterogenesis measured using 3H20 in liver, ileum and other extrahepatic tissues without affecting fatty acid synthesis. Compactin did not reduce serum or tissue cholesterol nor affect the serum concentration of other lipids nor the ratio between lipoprotein classes. A diurnal variation in the effect of compactin on cholesterogenesis was observed. For example, by 12--20 h after dosing, cholesterogenesis at all sites was increased above the comparable control value, indicating the induction of enzyme synthesis and overall there was little effect on the mass of cholesterol synthesized per day. Similar results were obtained using male chicks. Inhibition of cholesterogenesis by compactin was also observed in cholestyramine-treated rats, in which cholesterol turnover was markedly increased, and even in cholesterol-fed rats, in which cholesterogenesis already was repressed. In neither case, however, was inhibition of cholesterogenesis accompanied by a hypocholesterolaemic effect. It is concluded that a more persistent suppression of cholesterogenesis, than that observed with compactin in the rat, may be required in order to affect serum cholesterol concentrations.

  3. The Arabidopsis FLAKY POLLEN1 gene encodes a 3-hydroxy-3-methylglutaryl-coenzyme A synthase required for development of tapetum-specific organelles and fertility of pollen grains.

    PubMed

    Ishiguro, Sumie; Nishimori, Yuka; Yamada, Miho; Saito, Hiroko; Suzuki, Toshiya; Nakagawa, Tsuyoshi; Miyake, Hiroshi; Okada, Kiyotaka; Nakamura, Kenzo

    2010-06-01

    The pollen coat is a surface component of pollen grains required for fertilization. To study how the pollen coat is produced, we identified and characterized a recessive and conditional male-sterile Arabidopsis mutant, flaky pollen1-1 (fkp1-1), whose pollen grains lack functional pollen coats. FKP1 is a single-copy gene in the Arabidopsis genome and encodes 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMG-CoA synthase), an enzyme of the mevalonate (MVA) pathway involved in biosynthesis of isoprenoids such as sterols. We found that fkp1-1 possesses a T-DNA insertion 550 bp upstream of the initiation codon. RT-PCR and promoter analyses revealed that fkp1-1 results in knockdown of FKP1 predominantly in tapetum. Electron microscopy showed that the mutation affected the development of tapetum-specific lipid-containing organelles (elaioplast and tapetosome), causing the deficient formation of fkp1-1 pollen coats. These results suggest that both elaioplasts, which accumulate vast amount of sterol esters, and tapetosomes, which are unique oil-accumulating structures, require the MVA pathway for development. Null alleles of fkp1 were male-gametophyte lethal upon pollen tube elongation, whereas female gametophytes were normal. These results show that the MVA pathway is essential, at least in tapetal cells and pollen grains, for the development of tapetum-specific organelles and the fertility of pollen grains.

  4. Sterol regulatory element binding protein-mediated effect of fluvastatin on cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase transcription.

    PubMed

    Mascaró, C; Ortiz, J A; Ramos, M M; Haro, D; Hegardt, F G

    2000-02-15

    The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol.

  5. Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds.

    PubMed

    Forouhar, Farhad; Hussain, Munif; Farid, Ramy; Benach, Jordi; Abashidze, Mariam; Edstrom, William C; Vorobiev, Sergey M; Xiao, Rong; Acton, Thomas B; Fu, Zhuji; Kim, Jung-Ja P; Miziorko, Henry M; Montelione, Gaetano T; Hunt, John F

    2006-03-17

    The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can

  6. Crystal Structures of Two Bacterial 3-Hydroxy-3-methylglutaryl-CoA Lyases Suggest a Common Catalytic Mechanism among a Family of TIM Barrel Metalloenzymes Cleaving Carbon-Carbon Bonds

    SciTech Connect

    Forouhar,F.; Hussain, M.; Farid, R.; Benach, J.; Abashidze, M.; Edstrom, W.; Vorobiev, S.; Montelione, G.; Hunt, J.; et al.

    2006-01-01

    The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 {angstrom} resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name 'DRE-TIM metallolyases' for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and

  7. Statins in therapy: understanding their hydrophilicity, lipophilicity, binding to 3-hydroxy-3-methylglutaryl-CoA reductase, ability to cross the blood brain barrier and metabolic stability based on electrostatic molecular orbital studies.

    PubMed

    Fong, Clifford W

    2014-10-06

    The atomic electrostatic potentials calculated by the CHELPG method have been shown to be sensitive indicators of the gas phase and solution properties of the statins. Solvation free energies in water, n-octanol and n-octane have been determined using the SMD solvent model. The percentage hydrophilicity and hydrophobicity (or lipophilicity) of the statins in solution have been determined using (a) the differences in solvation free energies between n-octanol and n-octane as a measure of hydrophilicity, and the solvation energy in octane as a measure of hydrophobicity (b) the sum of the atomic electrostatic charges on the hydrogen bonding and polar bonding nuclei of the common pharmacophore combined with a solvent measure of hydrophobicity, and (c) using the buried surface areas after statin binding to HMGCR to calculate the hydrophobicity of the bound statins. The data suggests that clinical definitions of statins as either "hydrophilic" or "lipophilic" based on experimental partition coefficients are misleading. An estimate of the binding energy between rosuvastatin and HMGCR has been made using: (a) a coulombic electrostatic interaction model, (b) the calculated desolvation and resolvation of the statin in water, and (c) the first shell transfer solvation energy as a proxy for the restructuring of the water molecules immediately adjacent to the active binding site of HMGCR prior to binding. Desolvation and resolvation of the statins before and after binding to HMGCR are major determinants of the energetics of the binding process. An analysis of the amphiphilic nature of lovastatin anion, acid and lactone and fluvastatin anion and their abilities to cross the blood brain barrier has indicated that this process may be dominated by desolvation and resolvation effects, rather than the statin molecular size or statin-lipid interactions within the bilayer. The ionization energy and electron affinity of the statins are sensitive physical indicators of the ease that the various statins can undergo endogenous oxidative metabolism. The absolute chemical hardness is also an indicator of the stability of the statins, and may be a useful indicator for drug design.

  8. Statin (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor)-based therapy for hepatitis C virus (HCV) infection-related diseases in the era of direct-acting antiviral agents

    PubMed Central

    Kishta, Sara Sobhy; Kishta, Sobhy Ahmed; El-Shenawy, Reem

    2017-01-01

    Recent improvements have been made in the treatment of hepatitis C virus (HCV) infection with the introduction of direct-acting antiviral agents (DAAs). However, despite successful viral clearance, many patients continue to have HCV-related disease progression. Therefore, new treatments must be developed to achieve viral clearance and prevent the risk of HCV-related diseases. In particular, the use of pitavastatin together with DAAs may improve the antiviral efficacy as well as decrease the progression of liver fibrosis and the incidence of HCV-related hepatocellular carcinoma. To investigate the management methods for HCV-related diseases using pitavastatin and DAAs, clinical trials should be undertaken. However, concerns have been raised about potential drug interactions between statins and DAAs. Therefore, pre-clinical trials using a replicon system, human hepatocyte-like cells, human neurons and human cardiomyocytes from human-induced pluripotent stem cells should be conducted. Based on these pre-clinical trials, an optimal direct-acting antiviral agent could be selected for combination with pitavastatin and DAAs. Following the pre-clinical trial, the combination of pitavastatin and the optimal direct-acting antiviral agent should be compared to other combinations of DAAs ( e.g., sofosbuvir and velpatasvir) according to the antiviral effect on HCV infection, HCV-related diseases and cost-effectiveness. PMID:27583130

  9. Regulation of rat liver hydroxymethylglutaryl coenzyme A reductase by a new class of noncompetitive inhibitors. Effects of dichloroacetate and related carboxylic acids on enzyme activity.

    PubMed Central

    Stacpoole, P W; Harwood, H J; Varnado, C E

    1983-01-01

    Dichloroacetate (DCA) markedly reduces circulating cholesterol levels in animals and in patients with combined hyperlipoproteinemia or homozygous familial hypercholesterolemia (FH). To investigate the mechanism of its cholesterol-lowering action, we studied the effects of DCA and its hepatic metabolites, glyoxylate and oxalate, on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) obtained from livers of healthy, reverse light-cycled rats. Oral administration of DCA for 4 d decreased HMG CoA reductase activity 46% at a dose of 50 mg/kg per d, and 82% at a dose of 100 mg/kg per d. A 24% decrease in reductase activity was observed as early as 1 h after a single dose of 50 mg/kg DCA. The inhibitory effect of the drug was due to a fall in both expressed enzyme activity and the total number of reductase molecules present. DCA also decreased reductase activity when added to suspensions of isolated hepatocytes. With chronic administration, DCA inhibited 3H2O incorporation into cholesterol by 38% and into triglycerides by 52%. When liver microsomes were incubated with DCA, the pattern of inhibition of reductase activity was noncompetitive for both HMG CoA (inhibition constant [Ki] 11.8 mM) and NADPH (Ki 11.6 mM). Inhibition by glyoxylate was also noncompetitive for both HMG CoA (Ki 1.2 mM) and NADPH (Ki 2.7 mM). Oxalate inhibited enzyme activity only at nonsaturating concentrations of NADPH (Ki 5.6 mM). Monochloroacetate, glycollate, and ethylene glycol, all of which can form glyoxylate, also inhibited reductase activity. Using solubilized and 60-fold purified HMG CoA reductase, we found that the inhibitory effect of glyoxylate was reversible. Furthermore, the inhibition by glyoxylate was an effect exerted on the reductase itself, rather than on its regulatory enzymes, reductase kinase and reductase phosphatase. We conclude that the cholesterol-lowering effect of DCA is mediated, at least in part, by inhibition of endogenous cholesterol

  10. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj

    2011-03-01

    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

  11. Regulation of schistosome egg production by HMG CoA reductase

    SciTech Connect

    VandeWaa, E.A.; Bennett, J.L.

    1986-03-05

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of /sup 14/C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production.

  12. HMG-CoA reductase inhibitors decrease angiotensin II-induced vascular fibrosis: role of RhoA/ROCK and MAPK pathways.

    PubMed

    Rupérez, Mónica; Rodrigues-Díez, Raquel; Blanco-Colio, Luis Miguel; Sánchez-López, Elsa; Rodríguez-Vita, Juan; Esteban, Vanesa; Carvajal, Gisselle; Plaza, Juan José; Egido, Jesús; Ruiz-Ortega, Marta

    2007-08-01

    3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) present beneficial effects in cardiovascular diseases. Angiotensin II (Ang II) contributes to cardiovascular damage through the production of profibrotic factors, such as connective tissue growth factor (CTGF). Our aim was to investigate whether HMG-CoA reductase inhibitors could modulate Ang II responses, evaluating CTGF expression and the mechanisms underlying this process. In cultured vascular smooth muscle cells (VSMCs) atorvastatin and simvastatin inhibited Ang II-induced CTGF production. The inhibitory effect of statins on CTGF upregulation was reversed by mevalonate and geranylgeranylpyrophosphate, suggesting that RhoA inhibition could be involved in this process. In VSMCs, statins inhibited Ang II-induced Rho membrane localization and activation. In these cells Ang II regulated CTGF via RhoA/Rho kinase activation, as shown by inhibition of Rho with C3 exoenzyme, RhoA dominant-negative overexpression, and Rho kinase inhibition. Furthermore, activation of p38MAPK and JNK, and redox process were also involved in Ang II-mediated CTGF upregulation, and were downregulated by statins. In rats infused with Ang II (100 ng/kg per minute) for 2 weeks, treatment with atorvastatin (5 mg/kg per day) diminished aortic CTGF and Rho activation without blood pressure modification. Rho kinase inhibition decreased CTGF upregulation in rat aorta, mimicking statin effect. CTGF is a vascular fibrosis mediator. Statins diminished extracellular matrix (ECM) overexpression caused by Ang II in vivo and in vitro. In summary, HMG-CoA reductase inhibitors inhibit several intracellular signaling systems activated by Ang II (RhoA/Rho kinase and MAPK pathways and redox process) involved in the regulation of CTGF. Our results may explain, at least in part, some beneficial effects of statins in cardiovascular diseases.

  13. Research Finds Link Between Statin Use and Progressive Muscle Disease

    MedlinePlus

    ... guesses" led them to 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), an enzyme in the body ... Rosen LA. Autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase in patients with statin-associated autoimmune ...

  14. Conformational transitions of cinnamoyl CoA reductase 1 from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant D; Khan, Bashir M; Gaikwad, Sushama M

    2014-03-01

    Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.

  15. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.

  16. Genetics Home Reference: 3-hydroxy-3-methylglutaryl-CoA lyase deficiency

    MedlinePlus

    ... processed normally, a buildup of chemical byproducts called organic acids can result in metabolic acidosis. A shortage ... UK) FOD (Fatty Oxidation Disorders) Family Support Group Organic Acidemia Association Scientific Articles on PubMed (1 link) ...

  17. Impact of single-dose nandrolone decanoate on gonadotropins, blood lipids and HMG CoA reductase in healthy men.

    PubMed

    Gårevik, N; Börjesson, A; Choong, E; Ekström, L; Lehtihet, M

    2016-06-01

    The aim was to study the effect and time profile of a single dose of nandrolone decanoate (ND) on gonadotropins, blood lipids and HMG CoA reductase [3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)] in healthy men. Eleven healthy male participants aged 29-46 years were given a single dose of 150 mg ND as an intramuscular dose of Deca Durabol®, Organon. Blood samples for sex hormones, lipids and HMGCR mRNA analysis were collected prior to ND administration day 0, 4 and 14. A significant suppression of luteinising hormone (LH) and follicle-stimulating hormone (FSH) was seen after 4 days. Total testosterone and bioavailable testosterone level decreased significantly throughout the observed study period. A small but significant decrease in sexual hormone-binding globulin (SHBG) was seen after 4 days but not after 14 days. Total serum (S)-cholesterol and plasma (P)-apolipoprotein B (ApoB) increased significantly after 14 days. In 80% of the individuals, the HMGCR mRNA level was increased 4 days after the ND administration. Our results show that a single dose of 150 mg ND increases (1) HMGCR mRNA expression, (2) total S-cholesterol and (3) P-ApoB level. The long-term consequences on cardiovascular risk that may appear in users remain to be elucidated.

  18. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.

  19. Disposition of fluvastatin, an inhibitor of HMG-COA reductase, in mouse, rat, dog, and monkey.

    PubMed

    Tse, F L; Smith, H T; Ballard, F H; Nicoletti, J

    1990-01-01

    The physiological disposition of fluvastatin, a potent inhibitor of hydroxymethylglutaryl-CoA reductase and thus cholesterol synthesis, has been studied in the mouse, rat, dog, and monkey using 14C- or 3H-labeled drug. Oral doses of fluvastatin were absorbed at a moderate to rapid rate. The extent of absorption was dose-independent and was essentially complete in all four species studied. However, the drug was subject to extensive presystemic hepatic extraction followed by direct excretion via the bile, thus minimizing the systemic burden and yielding high liver/peripheral tissue concentration gradients for fluvastatin and its metabolites. Only at high doses far exceeding the intended human daily dose of ca 0.6 mg kg-1 did fluvastatin bioavailability approach unity, apparently due to saturation of the first-pass effect. Dose-normalized blood levels of fluvastatin and total radioactivity were higher in the dog than in the other species, suggesting a smaller distribution volume in the former. Fluvastatin was partially metabolized before excretion, the extent of metabolism being smallest in the dog and greatest in the mouse. The half-life of intact fluvastatin ranged from 1-2h in the monkey to 4-7h in the dog. Regardless of the dose or dose route, the administered radioactivity was recovered predominantly in feces, with the renal route accounting for less than 8 per cent of the dose. No tissue retention of radioactivity was observed, and material balance was essentially achieved within 96h after dosing.

  20. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  1. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    SciTech Connect

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  2. HMG-CoA reductase guides migrating primordial germ cells.

    PubMed

    Van Doren, M; Broihier, H T; Moore, L A; Lehmann, R

    1998-12-03

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is best known for catalysing a rate-limiting step in cholesterol biosynthesis, but it also participates in the production of a wide variety of other compounds. Some clinical benefits attributed to inhibitors of HMG-CoA reductase are now thought to be independent of any serum cholesterol-lowering effect. Here we describe a new cholesterol-independent role for HMG-CoA reductase, in regulating a developmental process: primordial germ cell migration. We show that in Drosophila this enzyme is highly expressed in the somatic gonad and that it is necessary for primordial germ cells to migrate to this tissue. Misexpression of HMG-CoA reductase is sufficient to attract primordial germ cells to tissues other than the gonadal mesoderm. We conclude that the regulated expression of HMG-CoA reductase has a critical developmental function in providing spatial information to guide migrating primordial germ cells.

  3. Can HMG Co-A reductase inhibitors (“statins”) slow the progression of age-related macular degeneration? The Age-Related Maculopathy Statin Study (ARMSS)

    PubMed Central

    Guymer, Robyn H; Dimitrov, Peter N; Varsamidis, Mary; Lim, Lyndell L; Baird, Paul N; Vingrys, Algis J; Robman, Luba

    2008-01-01

    Age-related macular degeneration (AMD) is responsible for the majority of visual impairment in the Western world. The role of cholesterol-lowering medications, HMG Co-A reductase inhibitors or statins, in reducing the risk of AMD or of delaying its progression has not been fully investigated. A 3-year prospective randomized controlled trial of 40 mg simvastatin per day compared to placebo in subjects at high risk of AMD progression is described. This paper outlines the primary aims of the Age-Related Maculopathy Statin Study (ARMSS), and the methodology involved. Standardized clinical grading of macular photographs and comparison of serial macular digital photographs, using the International grading scheme, form the basis for assessment of primary study outcomes. In addition, macular function is assessed at each visit with detailed psychophysical measurements of rod and cone function. Information collected in this study will assist in the assessment of the potential value of HMG Co-A reductase inhibitors (statins) in reducing the risk of AMD progression. PMID:18982929

  4. Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2014-05-01

    Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

  5. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis.

    PubMed

    Liu, Ying; Xu, Qiao-Xian; Xi, Pei-Yu; Chen, Hong-Hao; Liu, Chun-Sheng

    2013-05-01

    The roots of Glycyrrhiza uralensis are widely used in Chinese medicine for their action of clearing heat, detoxicating, relieving cough, dispelling sputum and tonifying spleen and stomach. The reason why Glycyrrhiza uralensis has potent and significant actions is that it contains various active secondary metabolites, especially glycyrrhizic acid. In the present study, we cloned the cDNA coding 3-hydroxy-3-methylglutary CoA reductase (HMGR) involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis. The corresponding cDNA was expressed in Escherichia coli as fusion proteins. Recombinant HMGR exhibited catalysis activity in reduction of HMG-CoA to mevalonic acid (MVA) just as HMGR isolated from other species. Because HMGR gene is very important in the biosynthesis of glycyrrhizic acid in Glycyrrhiza uralensis, this work is significant for further studies concerned with strengthening the efficacy of Glycyrrhiza uralensis by means of increasing glycyrrhizic acid content and exploring the biosynthesis of glycyrrhizic acid in vitro.

  6. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

  7. Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

    PubMed

    Srivastava, Sameer; Vishwakarma, Rishi K; Arafat, Yasir Ali; Gupta, Sushim K; Khan, Bashir M

    2015-04-01

    Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

  8. Incidence of Sepsis and Mortality With Prior Exposure of HMG-COA Reductase Inhibitors in a Surgical Intensive Care Population.

    PubMed

    Schurr, James W; Wu, Wenchen; Smith-Hannah, Alexandria; Smith, Candace J; Barrera, Rafael

    2016-01-01

    The anti-inflammatory properties of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may reduce the risk of developing sepsis in surgical intensive care patients and improve outcomes in those who do become septic. The objective of this study was to assess whether surgical intensive care unit (SICU) patients with prior exposure to HMG-CoA reductase inhibitors had a lower incidence of developing sepsis and improved outcomes. A retrospective cohort study was conducted. Patient demographic data, statin use, sequential organ failure assessment (SOFA) scores, vasopressor requirements, ventilator days, length of SICU stay, and mortality in septic patients were collected. Incidence of development of sepsis was determined using systemic inflammatory response syndrome criteria. Patients were grouped into cohorts based on whether they met the sepsis criteria and if they had previously received statins. Cohorts of patients who did and did not become septic with prior statin exposure were compared and an odds ratio was calculated to determine a protective effect. The setting was a SICU. The study comprised of 455 SICU patients and had no interventions. Among the 455 SICU patients, 427 patients were included for the final results. Patients receiving statins verses not receiving statins were similar in demographics. Previous statin exposure had a protective effect in the development of sepsis (9.77% on statins vs. 33.6% without statins; odds ratio 0.203, confidence interval 0.118-0.351). Of those patients who developed sepsis, there was a statistically significant decrease in 28-day mortality in patients with prior statin exposure (P = 0.0341). No statistical difference was noted in length of stay, vasopressor requirements, or days on mechanical ventilation. Prior exposure to statins may have a protective effect on the development of sepsis and decrease mortality in critically ill surgical patients.

  9. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull.) Persoon (Lion's Mane Mushroom)

    PubMed Central

    Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases. PMID:24959591

  10. Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

    PubMed

    Schwarz, Benjamin H; Driver, Joseph; Peacock, Riley B; Dembinski, Holly E; Corson, Melissa H; Gordon, Samuel S; Watson, Jeffrey M

    2014-02-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.

  11. HMG CoA reductase inhibitor-induced myotoxicity: pravastatin and lovastatin inhibit the geranylgeranylation of low-molecular-weight proteins in neonatal rat muscle cell culture.

    PubMed

    Flint, O P; Masters, B A; Gregg, R E; Durham, S K

    1997-07-01

    In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humans in vivo. In vitro myotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS-PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 microM lovastatin and 25 microM pravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 microM lovastatin and 400 microM pravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

  12. Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco.

    PubMed

    Prashant, S; Srilakshmi Sunita, M; Pramod, S; Gupta, Ranadheer K; Anil Kumar, S; Rao Karumanchi, S; Rawal, S K; Kavi Kishor, P B

    2011-12-01

    cDNA and genomic clones of cinnamoyl CoA reductase measuring 1011 and 2992 bp were isolated from a leguminous pulpwood tree Leucaena leucocephala, named as LlCCR. The cDNA exhibited 80-85% homology both at the nucleotide and amino acid levels with other known sequences. The genomic sequence contained five exons and four introns. Sense and antisense constructs of LlCCR were introduced in tobacco plants to up and down-regulate this key enzyme of lignification. The primary transformants showed a good correlation between CCR transcript levels and its activity. Most of the CCR down-regulated lines displayed stunted growth and development, wrinkled leaves and delayed senescence. These lines accumulated unusual phenolics like ferulic and sinapic acids in cell wall. Histochemical staining suggested reduction in aldehyde units and increased syringyl over guaiacyl (S/G) ratio of lignin. Anatomical studies showed thin walled, elongated xylem fibres, collapsed vessels with drastic reduction of secondary xylem. The transmission electron microscopic studies revealed modification of ultrastructure and topochemical distribution of wall polysaccharides and lignin in the xylem fibres. CCR down-regulated lines showed increased thickness of secondary wall layers and poor lignification of S2 and S3 wall layers. The severely down-regulated line AS17 exhibited 24.7% reduction of Klason lignin with an increase of 15% holocellulose content. Contrarily, the CCR up-regulated lines exhibited robust growth, development and significant increase in lignin content. The altered lignin profiles observed in transgenic tobacco lines support a role for CCR down-regulation in improving wood properties of L. leucocephala exclusively used in the pulp and paper industry of India.

  13. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

    PubMed

    Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

    2015-01-01

    Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

  14. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening

    PubMed Central

    Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

    2015-01-01

    Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening. PMID:26170618

  15. RNAi silencing of the HaHMG-CoA reductase gene inhibits oviposition in the Helicoverpa armigera cotton bollworm.

    PubMed

    Wang, Zhijian; Dong, Yongcheng; Desneux, Nicolas; Niu, Changying

    2013-01-01

    RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests.

  16. Effects of HMG-CoA reductase inhibitors (statins) on progression of kidney disease.

    PubMed

    Fried, Linda F

    2008-09-01

    Chronic kidney disease, especially in the setting of proteinuria, is characterized by hyperlipidemia. In animal models, hyperlipidemia causes glomerular foam cells and glomerulosclerosis. Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) ameliorates kidney disease in these models. The data of the role of hyperlipidemia in progression of human kidney disease are less clear. Data from small studies in glomerular disease suggest that statins decrease proteinuria. Data mainly from cardiovascular studies suggest that statins decrease the loss of glomerular filtration. The benefit of statins may derive from their lipid lowering effects. More recently, data suggest that the benefit of statins is greater than lipid lowering alone. The pleiotropic effects of statins may derive from inhibition of other downstream targets (isoprenoids) of the mevalonic acid pathway that are separate from cholesterol synthesis. Statins inhibits isoprenylation of Ras and Rho GTPases. These effects may lead to decreased monocyte/macrophage infiltration in the glomerulus, decreased mesangial proliferation and decreased accumulation of extracellular matrix and fibrosis. In addition, inhibition of RhoA and Ras may decrease inflammation and increase eNOS activity. These effects could lead to improvement in the progression of kidney disease.

  17. [Cloning and expression analysis of HMG-CoA reductase from Aquilaria sinensis (Lour.) Gilg].

    PubMed

    Xu, Yan-Hong; Yang, Xin; Zhang, Zheng; Liang, Liang; Wei, Jian-He

    2013-06-01

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate (MVA) pathway. The specific primers were designed according to the transcript sequence of AsHMGR2 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The full-length cDNA of AsHMGR2 was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) technology, and was analyzed at bioinformatics levels; AsHMGR2 expression profiles in different tissues and in responds to different treatments were analyzed by real-time PCR. The length of AsHMGR2 Open Reading Frame (ORF) was 1 749 bp, encoding 582 amino acids. The GenBank accession number is KC140287. Tissue expression analysis indicated that AsHMGR2 was mainly expressed in root and shoot tips, followed by stem, and was lowest in leaves. Inducible-experiments showed that the genes were induced by mechanical wound as well as chemical liquid induction, and reached the highest expression level at 6 h and 8 h, separately. The full-length cDNA of AsHMGR2 and its expression patterns will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

  18. Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors.

    PubMed

    Takeda, Michio; Noshiro, Rie; Onozato, Maristela Lika; Tojo, Akihiro; Hasannejad, Habib; Huang, Xiu-Lin; Narikawa, Shinichi; Endou, Hitoshi

    2004-01-12

    The purpose of this study was to elucidate the role of human organic anion transporters (human OATs) in the induction of drug-induced skeletal muscle abnormalities. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been clinically used for lowering plasma cholesterol levels, and are known to induce various forms of skeletal muscle abnormalities including myopathy and rhabdomyolysis. Immunohistochemical analysis revealed that human OAT1 and human OAT3 are localized in the cytoplasmic membrane of the human skeletal muscles. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs. Human OAT3, but not human OAT1, mediates the transport of pravastatin. Fluvastatin inhibited organic anion uptake mediated by human OAT1 in a mixture of competitive and noncompetitive manner, whereas simvastatin and fluvastatin noncompetitively inhibited the organic anion uptake mediated by human OAT3. In conclusion, the organic anion transporters OAT1 and OAT3 are localized in the cytoplasmic membrane of human skeletal muscles. Pravastatin, simvasatin, and fluvasatin inhibit human OATs activity. These results suggest that muscle organic anion transporters play a role in the muscular side effects of HMG-CoA reductase inhibitors.

  19. Inhibition of HMG-CoA reductase by MFS, a purified extract from the fermentation of marine fungus Fusarium solani FG319, and optimization of MFS production using response surface methodology.

    PubMed

    Zhou, Yu; Wu, Wen-Hui; Zhao, Qing-Bo; Wang, Xiao-Yu; Bao, Bin

    2015-05-01

    The present study was designed to isolate and characterize a purified extract from Fusarium solani FG319, termed MFS (Metabolite of Fusarium solani FG319) that showed anti-atherosclerosis activity by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Response surface methodology (RSM) was employed to achieve an improved yield from the fermentation medium. The inhibiting effect of the isolate, MFS, on HMG-CoA reductase was greater than that of the positive control, lovastatin. The average recovery of MFS and the relative standard deviation (RSD) ranged between 99.75% to 101.18%, and 0.31% to 0.74%, respectively. The RSDs intra- and inter-assay of the three samples ranged from 0.288% to 2.438%, and from 0.934% to 2.383%, respectively. From the RSM, the concentration of inducer, cultivation time, and culture temperatures had significant effects on the MFS production, with the effect of inducer concentration being more pronounced that other factors. In conclusion, the optimal conditions for the MFS production were achieved using RSM and that MFS could be explored as an anti-atherosclerosis agent based on its ability to inhibit HMG-CoA reductase.

  20. Myopathy induced by HMG-CoA reductase inhibitors in rabbits: a pathological, electrophysiological, and biochemical study.

    PubMed

    Nakahara, K; Kuriyama, M; Sonoda, Y; Yoshidome, H; Nakagawa, H; Fujiyama, J; Higuchi, I; Osame, M

    1998-09-01

    A combination of electrophysiological, pathological, and biochemical studies were performed in myopathy induced by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Simvastatin (a lipophilic inhibitor) or pravastatin (a hydrophilic inhibitor) were administered by gavage to rabbits. In Group I (simvastatin-treated group, 50 mg/kg/day for 4 weeks), four rabbits showed muscle necrosis and high serum creatine kinase (CK) levels, and all six rabbits showed electrical myotonia. In Group II (pravastatin-treated group, 100 mg/kg/day for 4 weeks), no rabbit showed either condition. In Group III (pravastatin-treated group, 200 mg/kg/day for 3 weeks plus 300 mg/kg/day for 3 weeks), one rabbit showed muscle necrosis and high serum CK level and two rabbits showed electrical myotonia. The pathological findings were muscle fiber necrosis and degeneration with increased acid phosphatase activity by light microscopy, autophagic vacuoles and mitochondrial swelling, and disruption and hypercontraction of myofibrils by electron microscopy. Ubiquinone content decreased in skeletal muscle by 22 to 36% in Group I, by 18 to 52% in Group II, and by 49 to 72% in Group III. However, mitochondrial enzyme activities of respiratory chain were normal in all groups. These results indicate that myopathy was not induced by a secondary dysfunction of mitochondrial respiration due to low ubiquinone levels.

  1. Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions.

    PubMed

    Trapani, Laura; Melli, Luca; Segatto, Marco; Trezza, Viviana; Campolongo, Patrizia; Jozwiak, Adam; Swiezewska, Ewa; Pucillo, Leopoldo Paolo; Moreno, Sandra; Fanelli, Francesca; Linari, Marco; Pallottini, Valentina

    2011-11-01

    The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

  2. Juxtamembranous aspartic acid in Insig-1 and Insig-2 is required for cholesterol homeostasis

    PubMed Central

    Gong, Yi; Lee, Joon No; Brown, Michael S.; Goldstein, Joseph L.; Ye, Jin

    2006-01-01

    Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum (ER) that mediate feedback control of cholesterol synthesis by sterol-dependent binding to the following two membrane proteins: the escort protein Scap, thus preventing proteolytic processing of sterol regulatory element-binding proteins; and the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase, thus inducing the ubiquitination and ER-associated degradation of the enzyme. Here, we report that the conserved Asp-205 in Insig-1, which abuts the fourth transmembrane helix at the cytosolic side of the ER membrane, is essential for its dual function. When Asp-205 was mutated to alanine, the mutant Insig-1 lost the ability to bind to Scap and, thus, was unable to suppress the cleavage of sterol regulatory element-binding proteins. The mutant Insig-1 was ineffective also in accelerating sterol-stimulated degradation of 3-hydroxy-3-methylglutaryl CoA reductase. Alanine substitution of the corresponding aspartic acid in Insig-2 produced the same dual defects. These studies identify a single amino acid residue that is crucial for the function of Insig proteins in regulating cholesterol homeostasis in mammalian cells. PMID:16606821

  3. Inhibition of M. tuberculosis β-ketoacyl CoA reductase FabG4 (Rv0242c) by triazole linked polyphenol-aminobenzene hybrids: comparison with the corresponding gallate counterparts.

    PubMed

    Banerjee, Deb Ranjan; Senapati, Kalyan; Biswas, Rupam; Das, Amit K; Basak, Amit

    2015-03-15

    Herein we report six novel triazole linked polyphenol-aminobenzene hybrids (3-8) as inhibitors of Mycobacterium tuberculosis FabG4 (Rv0242c), a less explored β-ketoacyl CoA reductase that has immense potential to be the future anti-tuberculosis drug target due to its possible involvement in drug resistance and latent infection. Novel triazole linked polyphenol-aminobenzene hybrids have been synthesized, characterized and evaluated for their inhibitory activity against FabG4. All of them inhibit FabG4 at low micromolar concentrations. In silico docking study has been carried out to explain the experimental findings. A comparative study of these new inhibitors with previously reported gallate counterparts leads to structure-activity relations (SAR) of substituent linked to N-1 of triazole ring.

  4. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

    PubMed

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control.

  5. HMG-CoA Reductase Inhibition Promotes Neurological Recovery, Peri-Lesional Tissue Remodeling, and Contralesional Pyramidal Tract Plasticity after Focal Cerebral Ischemia

    PubMed Central

    Kilic, Ertugrul; Reitmeir, Raluca; Kilic, Ülkan; Caglayan, Ahmet Burak; Beker, Mustafa Caglar; Kelestemur, Taha; Ethemoglu, Muhsine Sinem; Ozturk, Gurkan; Hermann, Dirk M.

    2014-01-01

    3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery. PMID:25565957

  6. Effects of HMG-CoA reductase inhibitors on the pharmacokinetics of losartan and its main metabolite EXP-3174 in rats: possible role of CYP3A4 and P-gp inhibition by HMG-CoA reductase inhibitors.

    PubMed

    Yang, Si-Hyung; Choi, Jun-Shik; Choi, Dong-Hyun

    2011-01-01

    The present study was designed to investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (atorvastatin, pravastatin, simvastatin) on the pharmacokinetics of losartan and its active metabolite EXP-3174 in rats. Pharmacokinetic parameters of losartan and EXP-3174 in rats were determined after oral and intravenous administration of losartan (9 mg/kg) without and with HMG-CoA reductase inhibitors (1 mg/kg). The effect of HMG-CoA reductase inhibitors on P-gp and cytochrome (CYP) 3A4 activity were also evaluated. Atorvastatin, pravastatin and simvastatin inhibited CYP3A4 activities with IC₅₀ values of 48.0, 14.1 and 3.10 μmol/l, respectively. Simvastatin (1-10 μmol/l) enhanced the cellular uptake of rhodamine-123 in a concentration-dependent manner. The area under the plasma concentration-time curve (AUC₀₋∞) and the peak plasma concentration of losartan were significantly (p < 0.05) increased by 59.6 and 45.8%, respectively, by simvastatin compared to those of control. The total body clearance (CL/F) of losartan after oral administration with simvastatin was significantly decreased (by 34.8%) compared to that of controls. Consequently, the absolute bioavailability (F) of losartan after oral administration with simvastatin was significantly increased by 59.4% compared to that of control. The metabolite-parent AUC ratio was significantly decreased by 25.7%, suggesting that metabolism of losartan was inhibited by simvastatin. In conclusion, the enhanced bioavailability of losartan might be mainly due to inhibition of P-gp in the small intestine and CYP3A subfamily-mediated metabolism of losartan in the small intestine and/or liver and to reduction of the CL/F of losartan by simvastatin.

  7. COAs: Behind the Masks.

    ERIC Educational Resources Information Center

    Birke, Szifra

    1993-01-01

    Provides information on alcoholism and codependency to help teachers identify and respond to children of alcoholics (COAs). Discusses characteristics of alcoholic homes and problems encountered by children and adult COAs. Examines survival "masks" of COAs, including hero, rebel, adjustor, clown, and caretaker. Lists organizational,…

  8. HMG-CoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase.

    PubMed

    Suh, Jung-Won; Choi, Dong-Ju; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-Soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10(-9)-10(-4) M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status.

  9. Camphene, a Plant-Derived Monoterpene, Reduces Plasma Cholesterol and Triglycerides in Hyperlipidemic Rats Independently of HMG-CoA Reductase Activity

    PubMed Central

    Vallianou, Ioanna; Peroulis, Nikolaos; Pantazis, Panayotis; Hadzopoulou-Cladaras, Margarita

    2011-01-01

    Background Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO). Methodology/Principal Findings The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001), 54% of Low Density Lipoprotein (LDL)-cholesterol (p<0.001) and 34.5% of triglycerides (p<0.001). Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. Conclusions Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent

  10. Potential risk of myopathy by HMG-CoA reductase inhibitors: a comparison of pravastatin and simvastatin effects on membrane electrical properties of rat skeletal muscle fibers.

    PubMed

    Pierno, S; De Luca, A; Tricarico, D; Roselli, A; Natuzzi, F; Ferrannini, E; Laico, M; Camerino, D C

    1995-12-01

    To get insight into the potential risk of myopathy associated with therapy involving 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, we evaluated in vivo and in vitro the effects of a daily 2 to 3-month treatment with pravastatin (100 mg/kg) and with simvastatin (5, 10 and 50 mg/kg) on the electrical properties of rat skeletal muscle fibers. The electromyographic activity revealed no sign of myopathy during treatment with pravastatin and with simvastatin. At the end of the treatment, the passive and active membrane electrical parameters of the extensor digitorum longus muscles were measured in vitro by computerized two-intracellular-microelectrode technique. A dose-dependent reduction of membrane chloride conductance was recorded in extensor digitorum longus fibers of simvastatin-treated groups, and at 50 mg/kg the reduction of chloride conductance was significant in 6 out of the 7 treated rats. By contrast, none of the pravastatin-treated rats showed significant alteration of chloride conductance. Consequently, the excitability parameters were modified by simvastatin but not by pravastatin treatment, whereas the resting membrane potential was not affected. An increase in potassium conductance, reduced by in vitro application of glybenclamide, was recorded in 30% of the simvastatin-treated rats (50 mg/kg) and in only 15% of the pravastatin-treated rats. Our results suggest that the risk of myopathy is much higher with the lipophilic simvastatin than with the hydrophilic pravastatin and support the hypothesis that the muscle toxicity of HMG-CoA reductase inhibitors is due to an intracellular action mediated by the inhibition of muscle cholesterol synthesis.

  11. [Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale].

    PubMed

    Zhang, Lin; Wang, Ji-Tao; Zhang, Da-Wei; Zhang, Gang; Guo, Shun-Xing

    2014-03-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

  12. Statins, HMG-CoA Reductase Inhibitors, Improve Neovascularization by Increasing the Expression Density of CXCR4 in Endothelial Progenitor Cells.

    PubMed

    Chiang, Kuang-Hsing; Cheng, Wan-Li; Shih, Chun-Ming; Lin, Yi-Wen; Tsao, Nai-Wen; Kao, Yung-Ta; Lin, Chih-Ting; Wu, Shinn-Chih; Huang, Chun-Yao; Lin, Feng-Yen

    2015-01-01

    Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, are used to reduce cholesterol biosynthesis in the liver. Accordingly, statins regulate nitric oxide (NO) and glutamate metabolism, inflammation, angiogenesis, immunity and endothelial progenitor cells (EPCs) functions. The function of EPCs are regulated by stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGF), and transforming growth factor β (TGF-β), etc. Even though the pharmacologic mechanisms by which statins affect the neovasculogenesis of circulating EPCs, it is still unknown whether statins affect the EPCs function through the regulation of CXCR4, a SDF-1 receptor expression. Therefore, we desired to explore the effects of statins on CXCR4 expression in EPC-mediated neovascularization by in vitro and in vivo analyses. In animal studies, we analyzed the effects of atorvastatin or rosuvastatin treatments in recovery of capillary density and blood flow, the expression of vWF and CXCR4 at ischemia sites in hindlimb ischemia ICR mice. Additionally, we analyzed whether the atorvastatin or rosuvastatin treatments increased the mobilization, homing, and CXCR4 expression of EPCs in hindlimb ischemia ICR mice that underwent bone marrow transplantation. The results indicated that statins treatment led to significantly more CXCR4-positive endothelial progenitor cells incorporated into ischemic sites and in the blood compared with control mice. In vivo, we isolated human EPCs and analyzed the effect of statins treatment on the vasculogenic ability of EPCs and the expression of CXCR4. Compared with the control groups, the neovascularization ability of EPCs was significantly improved in the atorvastatin or rosuvastatin group; this improvement was dependent on CXCR4 up-regulation. The efficacy of statins on improving EPC neovascularization was related to the SDF-1α/CXCR4 axis and might be regulated by the NO. In conclusion, atorvastatin and rosuvastatin improved

  13. Structure-based rational quest for potential novel inhibitors of human HMG-CoA reductase by combining CoMFA 3D QSAR modeling and virtual screening.

    PubMed

    Zhang, Qing Y; Wan, Jian; Xu, Xin; Yang, Guang F; Ren, Yan L; Liu, Jun J; Wang, Hui; Guo, Yu

    2007-01-01

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the formation of mevalonate. In many classes of organisms, this is the committed step leading to the synthesis of essential compounds, such as cholesterol. However, a high level of cholesterol is an important risk factor for coronary heart disease, for which an effective clinical treatment is to block HMGR using inhibitors like statins. Recently the structures of catalytic portion of human HMGR complexed with six different statins have been determined by a delicate crystallography study (Istvan and Deisenhofer Science 2001, 292, 1160-1164), which established a solid basis of structure and mechanism for the rational design, optimization, and development of even better HMGR inhibitors. In this study, three-dimensional quantitative structure-activity relationship (3D QSAR) with comparative molecular field analysis (CoMFA) was performed on a training set of up to 35 statins and statin-like compounds. Predictive models were established by using two different ways: (1) Models-fit, obtained by SYBYL conventional fit-atom molecular alignment rule, has cross-validated coefficients (q2) up to 0.652 and regression coefficients (r2) up to 0.977. (2) Models-dock, obtained by FlexE by docking compounds into the HMGR active site, has cross-validated coefficients (q2) up to 0.731 and regression coefficients (r2) up to 0.947. These models were further validated by an external testing set of 12 statins and statin-like compounds. Integrated with CoMFA 3D QSAR predictive models, molecular surface property (electrostatic and steric) mapping and structure-based (both ligand and receptor) virtual screening have been employed to explore potential novel hits for the HMGR inhibitors. A representative set of eight new compounds of non-statin-like structures but with high pIC(50) values were sorted out in the present study.

  14. Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors.

    PubMed

    Parker, R A; Clark, R W; Sit, S Y; Lanier, T L; Grosso, R A; Wright, J J

    1990-07-01

    Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also

  15. Suppressed production of methyl farnesoid hormones yields developmental defects and lethality in Drosophila larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A long-unresolved question in the developmental biology of Drosophila melanogaster has been whether methyl farnesoid hormones secreted by the ring gland are necessary for larval maturation and metamorphosis. In this study, we have used RNAi techniques to inhibit 3-Hydroxy-3-Methylglutaryl CoA Reduct...

  16. Developmental toxicity of the HMG-CoA reductase inhibitor (PPD10558) in rats and rabbits.

    PubMed

    Faqi, Ali S; Prohaska, David; Lopez, Rocio; McIntyre, Gail

    2012-02-01

    PPD10558 is an orally active, lipid-lowering 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin-associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0-24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect

  17. [Autoimmune myopathy associated with statin use].

    PubMed

    Ljøstad, Unn; Mygland, Åse

    2016-09-01

    It is well known that statins can have a toxic effect on musculature, but less widely known that they can also trigger progressive autoimmune myopathy. Statin-associated autoimmune myopathy is characterised by proximal muscle weakness, antibodies to 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) in serum, and necrosis without lymphocytic infiltration on muscle biopsy.

  18. Statin Drugs Markedly Inhibit Testosterone Production by Rat Leydig Cells In Vitro: Implications for Men

    EPA Science Inventory

    Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. During drug development it was shown that statins inhibit production of cholesterol in the testis. We evaluated testosterone production in vitro, using highly purified rat ...

  19. Pharmacodynamic potentiation of antiepileptic drugs' effects by some HMG-CoA reductase inhibitors against audiogenic seizures in DBA/2 mice.

    PubMed

    Russo, Emilio; Donato di Paola, Eugenio; Gareri, Pietro; Siniscalchi, Antonio; Labate, Angelo; Gallelli, Luca; Citraro, Rita; De Sarro, Giovambattista

    2013-04-01

    It is known that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are effective in both the primary and the secondary prevention of ischemic heart disease. Increasing evidence indicates that statins have protective effects in several neurological diseases including stroke, cerebral ischemia, Parkinson disease, multiple sclerosis, traumatic brain injury and epilepsy. The aim of the present research was to evaluate the effects of some HMG-CoA reductase inhibitors (i.e. lovastatin, simvastatin, atorvastatin, fluvastatin and pravastatin) commonly used for the treatment of hypercholesterolemia in the DBA/2 mice, an animal model of generalized tonic-clonic seizures. Furthermore, the co-administration of these compounds with some antiepileptic drugs (AEDs; i.e. carbamazepine, diazepam, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, topiramate and valproate) was studied in order to identify possible positive pharmacological interactions. Simvastatin only was active against both the tonic and clonic phase of audiogenic seizures, whereas the other statins tested were only partially effective against the tonic phase with the following order of potency: lovastatin>fluvastatin>atorvastatin; pravastatin was completely ineffective up to the dose of 150mg/kg. The co-administration of ineffective doses of all statins with AEDs generally increased the potency of the latter reducing their ED50 values. In particular, simvastatin was the most active in potentiating the activity of AEDs and the combinations of statins with carbamazepine, diazepam, felbamate, lamotrigine, topiramate and valproate were the most favorable, whereas, the co-administrations with the other AEDs studied was in most cases neutral. The increase in potency was generally associated with an enhancement of motor impairment (TD50); however, the therapeutic index (TD50/ED50) of combined treatment of AEDs with statins was predominantly more

  20. Platelet-derived growth factor stimulated mechanisms of glucosamine incorporation

    SciTech Connect

    Harrington, M.A.; Pledger, W.J. )

    1987-10-01

    Platelet-derived growth factor (PDGF) treatment of density-arrested BALB/c-3T3 cells results in increased ({sup 3}H)glucosamine (GlcN) incorporation into cellular material. The enhanced GlcN incorporation is not due to a preferential increase in proteoglycan synthesis as measured by ({sup 35}S)H{sub 2}SO{sub 4} incorporation. Approximately 50% of the GlcN incorporated in PDGF or platelet-poor plasma (PPP)-treated cultures enters N-linked glycoproteins. Addition of dolichol-phosphate (dolichol-P), a required intermediate in N-linked glycosylation, did not alter ({sup 3}H)GlcN incorporation in PDGF-treated cells but did increase incorporation in PPP-treated cultures to a level comparable to that observed for PDGF-treated cultures. PDGF-treated cultures contained twofold greater quantities of ({sup 3}H)GlcN dolichol intermediates and lipid-free glycoprotein. Over a 12-h time course 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity was similar in cultures treated with PDGF or PPP. Results of these studies reveal that enhanced protein glycosylation in response to PDGF treatment is not the result of a direct effect on HMG CoA reductase.

  1. Simvastatin, an HMG-CoA reductase inhibitor, induces the synthesis and secretion of apolipoprotein AI in HepG2 cells and primary hamster hepatocytes.

    PubMed

    Bonn, Victoria; Cheung, Raphael C; Chen, Biao; Taghibiglou, Changiz; Van Iderstine, Stephen C; Adeli, Khosrow

    2002-07-01

    Clinical studies have recently suggested that statin treatment may beneficially elevate plasma concentrations of high density lipoprotein (HDL)-cholesterol in patients with hyperlipidemia. Here, we have investigated the effect of a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase on the synthesis and secretion of apolipoprotein AI (apoAI) in two model systems, HepG2 cells and primary hamster hepatocytes. Cultured cells were incubated with different doses of simvastatin (0.1-10 microM) for a period of 18 h. A dose-dependent increase in synthesis and secretion of apoAI was observed in both cell types. There was a significant increase in the synthesis of apoAI in HepG2 cells (44.3+/-12.1%), and hamster hepatocytes (212+/-2%) after treatment with 10 microM of the statin. The increase in apoAI synthesis appeared to result in a higher level of apoAI secreted into the culture media in both cell types (49.2+/-7.8% in HepG2, 197+/-0.2% in hamster hepatocytes). ApoAI mRNA levels were also significantly increased in both cell types in response to statin treatment. Control experiments with transferrin confirmed specificity of the effect on apoAI secretion. Analysis of a density fraction containing HDL particles in culture media revealed an increase in HDL-associated apoAI of 94.3+/-2.1% in HepG2 cells and 27.0+/-0.03% in hamster hepatocytes following 10 microM simvastatin-treatment. Comparative studies of simvastatin and lovastatin indicated a differential ability to induce apoAI synthesis and secretion, with simvastatin having a more significant effect. Thus, acute statin treatment of cultured hepatocytes (transformed as well as primary) resulted in a significant upregulation of apoAI mRNA and apoAI synthesis, causing oversecretion of apoAI and HDL extracellularly. The stimulatory effect on apoAI synthesis and secretion may thus explain the clinical observation of an elevated plasma HDL-cholesterol level in hyperlipidemic patients treated with

  2. Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis.

    PubMed

    Dias, Marcio Vinicius Bertacine; Vasconcelos, Igor Bordin; Prado, Adriane Michele Xavier; Fadel, Valmir; Basso, Luiz Augusto; de Azevedo, Walter Filgueira; Santos, Diógenes Santiago

    2007-09-01

    The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

  3. Efficacy, tissue distribution and biliary excretion of methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate (CP-83101), a hepatoselective inhibitor of HMG-CoA reductase activity in the rat.

    PubMed

    Harwood, H J; Silva, M; Chandler, C E; Mikolay, L; Pellarin, L D; Barbacci-Tobin, E; Wint, L T; McCarthy, P A

    1990-09-15

    Methyl (3R*,5S*)-(E)-3,5-dihydroxy-9,9-diphenyl-6,8-nonadienoate, CP-83101, was identified as a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, inhibiting enzyme activity in vitro with an IC50 of 8.5 +/- 0.7 microM and a Ki with respect to HMG-CoA of 2.6 microM. CP-83101 also inhibited rat hepatic sterol biosynthesis by 39 +/- 7% at a dose of 100 mg/kg. [3H]CP-83101, administered orally to rats, exhibited peak plasma levels at approximately 1 hr that declined thereafter with an apparent half-time of 2-3 hr. Peak tissue levels also occurred 1 hr following oral administration of [3H]CP-83101. The decline in radioactivity in the liver, however, was considerably slower than that noted in blood, whereas the half-life in non-hepatic tissues was approximately 1 hr. Liver/blood ratios of 14, and liver/lens ratios of greater than 3000, following oral administration of [3H]CP-83101, were similar to those previously reported for other HMG-CoA reductase inhibitors, suggesting a high degree of tissue selectivity. In addition, liver/adrenal and liver/ovary ratios were approximately 1000 at all time points examined between 30 min and 24 hr following oral [3H]CP-83101 administration, indicating a high specificity for hepatic versus other steroidogenic tissues. Evaluation of intravenous versus oral administration of the water-soluble, free acid, sodium salt of [3H]CP-83101 in bile duct canulated rats indicated that approximately 20% of orally administered CP-83101 is absorbed from the gastrointestinal tract, and that absorbed CP-83101 is cleared rapidly from the plasma via the liver and from the liver via the bile. In addition, several lines of evidence suggest that CP-83101 may undergo enterohepatic recirculation. Agents of this synthetic series may thus possess advantages over other HMG-CoA reductase inhibitors with respect to tissue kinetics and specificity.

  4. Effects of manufactured soluble dietary fiber from Quercus mongolica on hepatic HMG-CoA reductase and lipoprotein lipase activities in epididymal adipose tissue of rats fed high cholesterol diets.

    PubMed

    Chai, Young-Mi; Lim, Bu-Kug; Lee, Jong-Yoon; Kim, Mu-Nam; Park, Mo-Ra; Rhee, Soon-Jae

    2003-01-01

    This study investigated the effect of a manufactured soluble dietary fiber on lipid metabolism in rats fed high cholesterol diets. Soluble dietary fiber was prepared from wood chips of oak (Quercus mongolica). Male Sprague-Dawley rats weighing 100 +/- 10 g were randomly assigned to either a normal diet or five high cholesterol diets containing 1% cholesterol and different fiber supplements. The high cholesterol groups were subdivided into fiber-free diet (FF), 5% pectin (5P), 10% pectin (10P), 5% manufactured soluble dietary fiber (5QM), and 10% manufactured soluble dietary fiber (10QM) groups. Total serum cholesterol concentrations in all soluble dietary fiber-supplemented groups were lower than in the FF group. The high-density lipoprotein-cholesterol concentration in the FF group was significantly lower, compared with the normal group, but was increased in groups supplemented with soluble dietary fiber. Low-density lipoprotein-cholesterol levels and the atherogenic index had the same tendency as total cholesterol concentration. Compared with the FF group, in the 5P, 5QM, 10P, and 10QM groups hepatic triglyceride concentrations were 12%, 16%, 20%, and 24% lower, respectively, and hepatic cholesterol concentrations were 48%, 52%, 52%, and 58% lower, respectively. Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity in the soluble fiber groups was significantly higher than in the FF groups, but lower than the normal group. When hepatic tissue was observed under a light microscope, the FF group had completely formed lipomas in the hepatic tissue, which led to fat deposits and then a fatty liver. The size and number of lipomas were lower in the soluble dietary fiber-fed groups, as compared with the group not fed dietary fiber. In conclusion, improvements in lipid metabolism were observed as a result of the manufactured soluble dietary fiber from the oak chips, and were similar to that seen for pectin. The preparation method for the soluble dietary fiber from oak

  5. Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

    PubMed

    Kocarek, T A; Reddy, A B

    1996-11-01

    It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of

  6. Potential of tocotrienols in the prevention and therapy of Alzheimer's disease.

    PubMed

    Xia, Weiming; Mo, Huanbiao

    2016-05-01

    Currently there is no cure for Alzheimer's disease (AD); clinical trials are underway to reduce amyloid generation and deposition, a neuropathological hallmark in brains of AD patients. While genetic factors and neuroinflammation contribute significantly to AD pathogenesis, whether increased cholesterol level is a causative factor or a result of AD is equivocal. Prenylation of proteins regulating neuronal functions requires mevalonate-derived farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The observation that the levels of FPP and GGPP, but not that of cholesterol, are elevated in AD patients is consistent with the finding that statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, reduce FPP and GGPP levels and amyloid β protein production in preclinical studies. Retrospective studies show inverse correlations between incidence of AD and the intake and serum levels of the HMG CoA reductase-suppressive tocotrienols; tocopherols show mixed results. Tocotrienols, but not tocopherols, block the processing and nuclear localization of sterol regulatory element binding protein-2, the transcriptional factor for HMG CoA reductase and FPP synthase, and enhance the degradation of HMG CoA reductase. Consequently, tocotrienols deplete the pool of FPP and GGPP and potentially blunt prenylation-dependent AD pathogenesis. The antiinflammatory activity of tocotrienols further contributes to their protection against AD. The mevalonate- and inflammation-suppressive activities of tocotrienols may represent those of an estimated 23,000 mevalonate-derived plant secondary metabolites called isoprenoids, many of which are neuroprotective. Tocotrienol-containing plant foods and tocotrienol derivatives and formulations with enhanced bioavailability may offer a novel approach in AD prevention and treatment.

  7. A Combination of Flaxseed Oil and Astaxanthin Improves Hepatic Lipid Accumulation and Reduces Oxidative Stress in High Fat-Diet Fed Rats

    PubMed Central

    Xu, Jiqu; Rong, Shuang; Gao, Hui; Chen, Chang; Yang, Wei; Deng, Qianchun; Huang, Qingde; Xiao, Lingyun; Huang, Fenghong

    2017-01-01

    Hepatic lipid accumulation and oxidative stress are crucial pathophysiological mechanisms for non-alcoholic fatty liver disease (NAFLD). Thus, we examined the effect of a combination of flaxseed oil (FO) and astaxanthin (ASX) on hepatic lipid accumulation and oxidative stress in rats fed a high-fat diet. ASX was dissolved in flaxseed oil (1 g/kg; FO + ASX). Animals were fed diets containing 20% fat, where the source was lard, or 75% lard and 25% FO + ASX, or 50% lard and 50% FO + ASX, or FO + ASX, for 10 weeks. Substitution of lard with FO + ASX reduced steatosis and reduced hepatic triacylglycerol and cholesterol. The combination of FO and ASX significantly decreased hepatic sterol regulatory element-binding transcription factor 1 and 3-hydroxy-3-methylglutaryl-CoA reductase but increased peroxisome proliferator activated receptor expression. FO + ASX significantly suppressed fatty acid synthase and acetyl CoA carboxylase but induced carnitine palmitoyl transferase-1 and acyl CoA oxidase expression. FO + ASX also significantly elevated hepatic SOD, CAT and GPx activity and GSH, and markedly reduced hepatic lipid peroxidation. Thus, FO and ASX may reduce NAFLD by reversing hepatic steatosis and reducing lipid accumulation and oxidative stress. PMID:28335388

  8. Identifying statin-associated autoimmune necrotizing myopathy.

    PubMed

    Albayda, Jemima; Christopher-Stine, Lisa

    2014-12-01

    Statins up-regulate expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis and the major target of autoantibodies in statin-associated immune-mediated necrotizing myopathy. As muscle cells regenerate, they express high levels of HMGCR, which may sustain the immune response even after statin therapy is stopped. Awareness of this entity will help physicians who prescribe statins to take action to limit the associated morbidity.

  9. Peroxisome proliferator-activated receptor γ down-regulation mediates the inhibitory effect of d-δ-tocotrienol on the differentiation of murine 3T3-F442A preadipocytes.

    PubMed

    Torabi, Sheida; Yeganehjoo, Hoda; Shen, Chwan-Li; Mo, Huanbiao

    2016-12-01

    Tocotrienols accelerate the degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase that catalyzes the biosynthesis of mevalonate; the latter is essential for preadipocyte differentiation. Tocotrienols also down-regulate peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation. We hypothesized that mevalonate deprivation and PPARγ down-regulation mediate d-δ-tocotrienol-induced inhibition of adipocyte differentiation. The objectives of this study were to determine the effect of d-δ-tocotrienol on 3T3-F442A preadipocyte differentiation and the involvement of PPARγ and mevalonate. Murine 3T3-F442A preadipocytes were incubated with d-δ-tocotrienol (2.5-10 μmol/L) for 8 days. AdipoRed assay and Oil Red O staining showed that d-δ-tocotrienol dose-dependently reduced the intracellular triglyceride content. Concomitantly, d-δ-tocotrienol dose-dependently inhibited glucose uptake by 3T3-F442A cells and the expression of GLUT4, HMG CoA reductase, and p-Akt proteins. The effects of d-δ-tocotrienol on intracellular triglyceride content and glucose uptake were attenuated by rosiglitazone, an agonist of PPARγ, but not supplemental mevalonate (100 μmol/L). In contrast, mevalonate, but not rosiglitazone, reversed the effects of lovastatin, a competitive inhibitor of HMG CoA reductase shown to inhibit adipocyte differentiation via mevalonate deprivation. Trypan blue staining revealed no changes in cell viability after a 48-hour incubation of 3T3-F442A cells with d-δ-tocotrienol (0-80 μmol/L), suggesting that the adipogenesis-suppressive activity of d-δ-tocotrienol was independent of cytotoxicity. In conclusion, these findings demonstrate the antiadipogenic effect of d-δ-tocotrienol via PPARγ down-regulation.

  10. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight: evidence from genetic analysis and randomised trials

    PubMed Central

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B; Holmes, Michael V; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, KaWah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A; Drenos, Fotios; Li, Yun R; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G; van der A, Daphne L; Forouhi, Nita G; Onland-Moret, N Charlotte; van der Schouw, Yvonne T; Schnabel, Renate B; Hubacek, Jaroslav A; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J Wouter; Westendorp, Rudi G J; de Borst, Gert Jan; de Jong, Pim A; Algra, Ale; Spiering, Wilko; der Zee, Anke H Maitland-van; Klungel, Olaf H; de Boer, Anthonius; Doevendans, Pieter A; Eaton, Charles B; Robinson, Jennifer G; Duggan, David; Kjekshus, John; Downs, John R; Gotto, Antonio M; Keech, Anthony C; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S; Poulter, Neil R; Waters, David D; Pedersen, Terje R; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D; Chasman, Daniel I; Ridker, Paul M; Maggioni, Aldo P; Tavazzi, Luigi; Ray, Kausik K; Seshasai, Sreenivasa Rao Kondapally; Manson, JoAnn E; Price, Jackie F; Whincup, Peter H; Morris, Richard W; Lawlor, Debbie A; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J; Fornage, Myriam; Siscovick, David S; Cushman, Mary; Kumari, Meena; Wareham, Nick J; Verschuren, W M Monique; Redline, Susan; Patel, Sanjay R; Whittaker, John C; Hamsten, Anders; Delaney, Joseph A; Dale, Caroline; Gaunt, Tom R; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A; van der Harst, Pim; Brunner, Eric J; Tybjaerg-Hansen, Anne; Marmot, Michael G; Krauss, Ronald M; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C; Psaty, Bruce M; Lange, Leslie A; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E; Talmud, Philippa J; Kivimäki, Mika; Timpson, Nicholas J; Langenberg, Claudia; Asselbergs, Folkert W; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G; Reiner, Alex P; Keating, Brendan J; Hingorani, Aroon D; Sattar, Naveed

    2015-01-01

    Summary Background Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. Methods We used single nucleotide polymorphisms in the HMGCR gene, rs17238484 (for the main analysis) and rs12916 (for a subsidiary analysis) as proxies for HMGCR inhibition by statins. We examined associations of these variants with plasma lipid, glucose, and insulin concentrations; bodyweight; waist circumference; and prevalent and incident type 2 diabetes. Study-specific effect estimates per copy of each LDL-lowering allele were pooled by meta-analysis. These findings were compared with a meta-analysis of new-onset type 2 diabetes and bodyweight change data from randomised trials of statin drugs. The effects of statins in each randomised trial were assessed using meta-analysis. Findings Data were available for up to 223 463 individuals from 43 genetic studies. Each additional rs17238484-G allele was associated with a mean 0·06 mmol/L (95% CI 0·05–0·07) lower LDL cholesterol and higher body weight (0·30 kg, 0·18–0·43), waist circumference (0·32 cm, 0·16–0·47), plasma insulin concentration (1·62%, 0·53–2·72), and plasma glucose concentration (0·23%, 0·02–0·44). The rs12916 SNP had similar effects on LDL cholesterol, bodyweight, and waist circumference. The rs17238484-G allele seemed to be associated with higher risk of type 2 diabetes (odds ratio [OR] per allele 1·02, 95% CI 1·00–1·05); the rs12916-T allele association was consistent (1·06, 1·03–1·09). In 129 170 individuals in randomised trials, statins lowered LDL cholesterol by 0·92 mmol/L (95% CI 0·18–1·67) at 1-year of follow-up, increased bodyweight by 0·24 kg (95% CI 0·10–0·38 in all trials; 0·33 kg, 95% CI 0·24–0·42 in placebo or standard care controlled trials and −0·15 kg, 95% CI −0·39 to 0·08 in intensive

  11. Possible mechanisms underlying statin-induced skeletal muscle toxicity in L6 fibroblasts and in rats.

    PubMed

    Itagaki, Mai; Takaguri, Akira; Kano, Seiichiro; Kaneta, Shigeru; Ichihara, Kazuo; Satoh, Kumi

    2009-01-01

    3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are safe and well-tolerated therapeutic drugs. However, they occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Here, we investigated the mechanism of statin-induced myotoxicity in L6 fibroblasts and in rats in vivo. L6 fibroblasts were differentiated and then treated with pravastatin, simvastatin, or fluvastatin for 72 h. Hydrophobic simvastatin and fluvastatin decreased cell viability in a dose-dependent manner via apoptosis characterized by typical nuclear fragmentation and condensation and caspase-3 activation. Both hydrophobic statins transferred RhoA localization from the cell membrane to the cytosol. These changes induced by both hydrophobic statins were completely abolished by the co-application of geranylgeranylpyrophosphate (GGPP). Y27632, a Rho-kinase inhibitor, mimicked the hydrophobic statin-induced apoptosis. Hydrophilic pravastatin did not affect the viability of the cells. Fluvastatin was continuously infused (2.08 mg/kg at an infusion rate of 0.5 mL/h) into the right internal jugular vein of the rats in vivo for 72 h. Fluvastatin infusion significantly elevated the plasma CPK level and transferred RhoA localization in the skeletal muscle from the cell membrane to the cytosol. In conclusion, RhoA dysfunction due to loss of lipid modification with GGPP is involved in the mechanisms of statin-induced skeletal muscle toxicity.

  12. A role for the mevalonate pathway in early plant symbiotic signaling

    PubMed Central

    Venkateshwaran, Muthusubramanian; Jayaraman, Dhileepkumar; Chabaud, Mireille; Genre, Andrea; Balloon, Allison J.; Maeda, Junko; Forshey, Kari; den Os, Désirée; Kwiecien, Nicholas W.; Coon, Joshua J.; Barker, David G.; Ané, Jean-Michel

    2015-01-01

    Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca2+ concentration (Ca2+ spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca2+ spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume–rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca2+ spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca2+ spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca2+ spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca2+ spiking in this heterologous system. PMID:26199419

  13. Cooked rice prevents hyperlipidemia in hamsters fed a high-fat/cholesterol diet by the regulation of the expression of hepatic genes involved in lipid metabolism.

    PubMed

    Choi, Won Hee; Gwon, So Young; Ahn, Jiyun; Jung, Chang Hwa; Ha, Tae Youl

    2013-07-01

    Rice has many health-beneficial components for ameliorating obesity, diabetes, and dyslipidemia. However, the effect of cooked rice as a useful carbohydrate source has not been investigated yet; so we hypothesized that cooked rice may have hypolipidemic effects. In the present study, we investigated the effect of cooked rice on hyperlipidemia and on the expression of hepatic genes involved in lipid metabolism. Golden Syrian hamsters were divided into 2 groups and fed a high-fat (15%, wt/wt)/cholesterol (0.5%, wt/wt) diet supplemented with either corn starch (HFD, 54.5% wt/wt) or cooked rice (HFD-CR, 54.5% wt/wt) as the main carbohydrate source for 8 weeks. In the HFD-CR group, the triglyceride and total cholesterol levels in the serum and liver were decreased, and the total lipid, total cholesterol, and bile acid levels in the feces were increased, compared with the HFD group. In the cooked-rice group, the messenger RNA and protein levels of 3-hydroxy-3-methylglutaryl CoA reductase were significantly downregulated; and the messenger RNA and protein levels of the low-density lipoprotein receptor and cholesterol-7α-hydroxylase were upregulated. Furthermore, the expressions of lipogenic genes such as sterol response element binding protein-1, fatty acid synthase, acetyl CoA carboxylase, and stearoyl CoA desaturase-1 were downregulated, whereas the β-oxidation related genes (carnitine palmitoyl transferase-1, acyl CoA oxidase, and peroxisome proliferator-activated receptor α) were upregulated, in the cooked-rice group. Our results suggest that the hypolipidemic effect of cooked rice is partially mediated by the regulation of hepatic genes involved in lipid metabolism, which results in the suppression of cholesterol and fatty acid synthesis and the enhancement of cholesterol excretion and fatty acid β-oxidation.

  14. Cholesterol biosynthesis regulation and protein changes in rat liver following treatment with fluvastatin.

    PubMed

    Steiner, S; Gatlin, C L; Lennon, J J; McGrath, A M; Seonarain, M D; Makusky, A J; Aponte, A M; Esquer-Blasco, R; Anderson, N L

    2001-03-31

    The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulator in cholesterol biosynthesis and HMG CoA reductase inhibitors (statins) have become a widely prescribed family of lipid lowering agents. Cholesterol synthesis occurs predominantly in liver which is the target organ of statins. We studied the effects of fluvastatin (Lescol), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P<0.005). Major effects were evident in the cholesterol biosynthesis pathway including the induction of enzymes upstream and downstream of the target enzyme HMG CoA reductase. Treatment also triggered alterations in key enzymes of carbohydrate metabolism and was associated with changes in a heterogeneous set of cellular stress proteins involved in cytoskeletal structure, calcium homeostasis and protease activity. The latter set of protein alterations indicates that hepatotoxicity is associated with high-dose treatment. Based on the results it is suggested that HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase may be explored as alternative drug targets and that the induction levels of these enzymes may serve as a measure of potency of individual statin drugs. It is proposed that efficacy and cellular stress markers discovered in this study may be used in a high throughput screen (HTS) assay format to compare efficiently and accurately the therapeutic windows of different members of the statin family.

  15. Antihyperlipidemic and Antioxidant Effects of C-phycocyanin in Golden Syrian Hamsters Fed with a Hypercholesterolemic Diet.

    PubMed

    Sheu, Ming-Jyh; Hsieh, Yao-Yuan; Lai, Ching-Hsiu; Chang, Chi-Chen; Wu, Chieh-Hsi

    2013-01-01

    Hyperlipidemia and oxidation play major roles upon cardiovascular diseases (CVDs). C-phycocyanin (CPC), the major component in blue-green algae, possesses antiinflammatory and radical scavenging properties. Herein we aimed to investigate the effect of CPC upon lipid metabolism and its antioxidant effects. Golden Syrian hamsters were randomly assigned to five groups: (1) control; (2) 0.2% cholesterol; (3) 0.2% cholesterol+ 1% lopid; (4) 0.2% cholesterol+ 0.25% CPC; and (5) 0.2% cholesterol+ 1.25% CPC. All animals were sacrificed after 8-week feeding. Serum cholesterol, triglyceride (TG), low-density lipoprotein (LDL), glutamate-oxaloacetate transaminase (GOT), and glutamate-pyruvate transaminase (GPT) were examined. The diene conjugation in the Cu(2+)-mediated oxidation of LDL was measured. The protein levels of several antioxidative enzymes including catalase (CAT), superoxide dismutases (SOD), and glutathione peroxidase (GPx) of liver were assayed. HepG2 cells were cultured in medium containing various concentrations of CPC (0, 1, 15, and 30 μM). The mRNA concentrations of LDL receptor, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, SOD-1 and GPx of HepG2 cells in each group were analyzed. CPC was effective in lowering serum cholesterol, total cholesterol (TC), TG, LDL, GOT, and GPT. CPC was found to decrease the malondialdehyde (MDA) equivalents and delay the diene conjugation in the Cu(2+)-mediated oxidation of LDL. CPC increase the enzyme expressions of CAT, SOD, and GPx. CPC concentrations were positively correlated with the mRNA level of LDL receptor while the mRNA levels of HMG CoA reductase, SOD-1, and GPx in HepG2 cells were not affected. The lipid-lowering and antioxidation effects of CPC suggest its roles in prevention of CVD and atherosclerotic formation.

  16. Effect of dietary n-3 fatty acids supplementation on fatty acid metabolism in atorvastatin-administered SHR.Cg-Lepr(cp)/NDmcr rats, a metabolic syndrome model.

    PubMed

    Al Mamun, Abdullah; Hashimoto, Michio; Katakura, Masanori; Tanabe, Yoko; Tsuchikura, Satoru; Hossain, Shahdat; Shido, Osamu

    2017-01-01

    The effects of cholesterol-lowering statins, which substantially benefit future cardiovascular events, on fatty acid metabolism have remained largely obscured. In this study, we investigated the effects of atorvastatin on fatty acid metabolism together with the effects of TAK-085 containing highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl ester on atorvastatin-induced n-3 polyunsaturated fatty acid lowering in SHR.Cg-Lepr(cp)/NDmcr (SHRcp) rats, as a metabolic syndrome model. Supplementation with 10mg/kg body weight/day of atorvastatin for 17 weeks significantly decreased plasma total cholesterol and very low density lipoprotein cholesterol. Atorvastatin alone caused a subtle change in fatty acid composition particularly of EPA and DHA in the plasma, liver or erythrocyte membranes. However, the TAK-085 consistently increased both the levels of EPA and DHA in the plasma, liver and erythrocyte membranes. After confirming the reduction of plasma total cholesterol, 300mg/kg body weight/day of TAK-085 was continuously administered for another 6 weeks. Supplementation with TAK-085 did not decrease plasma total cholesterol but significantly increased the EPA and DHA levels in both the plasma and liver compared with rats administered atorvastatin only. Supplementation with atorvastatin alone significantly decreased sterol regulatory element-binding protein-1c, Δ5- and Δ6-desaturases, elongase-5, and stearoyl-coenzyme A (CoA) desaturase-2 levels and increased 3-hydroxy-3-methylglutaryl-CoA reductase mRNA expression in the liver compared with control rats. TAK-085 supplementation significantly increased stearoyl-CoA desaturase-2 mRNA expression. These results suggest that long-term supplementation with atorvastatin decreases the EPA and DHA levels by inhibiting the desaturation and elongation of n-3 fatty acid metabolism, while TAK-085 supplementation effectively replenishes this effect in SHRcp rat liver.

  17. Regulation of hepatic 7 alpha-hydroxylase expression by dietary psyllium in the hamster.

    PubMed Central

    Horton, J D; Cuthbert, J A; Spady, D K

    1994-01-01

    Soluble fiber consistently lowers plasma total and low density lipoprotein (LDL)-cholesterol concentrations in humans and various animal models including the hamster; however, the mechanism of this effect remains incompletely defined. We performed studies to determine the activity of dietary psyllium on hepatic 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and LDL receptor expression in the hamster. In animals fed a cholesterol-free semisynthetic diet containing 7.5% cellulose (avicel) as a fiber source, substitution of psyllium for avicel increased hepatic 7 alpha-hydroxylase activity and mRNA levels by 3-4-fold. Comparable effects on 7 alpha-hydroxylase expression were observed with 1% cholestyramine. Psyllium also increased hepatic 7 alpha-hydroxylase activity and mRNA in animals fed a diet enriched with cholesterol and triglyceride. Activation of 7 alpha-hydroxylase was associated with an increase in hepatic cholesterol synthesis that was apparently not fully compensatory since the cholesterol content of the liver declined. Although dietary psyllium did not increase hepatic LDL receptor expression in animals fed the cholesterol-free, very-low-fat diet, it did increase (or at least restore) receptor expression that had been downregulated by dietary cholesterol and triglyceride. Thus, 7.5% dietary psyllium produced effects on hepatic 7 alpha-hydroxylase and LDL metabolism that were similar to those of 1% cholestyramine. Induction of hepatic 7 alpha-hydroxylase activity by dietary psyllium may account, in large part, for the hypocholesterolemic effect of this soluble fiber. Images PMID:8182140

  18. Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes.

    PubMed

    Coward, William R; Marei, Ayman; Yang, AiLi; Vasa-Nicotera, Mariuca M; Chow, Sek C

    2006-05-01

    Statins, which inhibit 3-hydroxy-3-methylglutaryl CoA reductase, have been shown recently to promote proinflammatory responses. We show in this study that both atorvastatin and simvastatin induced proinflammatory responses in mitogen-activated PBMCs by increasing the number of T cells secreting IFN-gamma. This is abolished by the presence of mevalonate, suggesting that statins act specifically by blocking the mevalonate pathway for cholesterol synthesis to promote the proinflammatory response. Both statins at low concentrations induced a dose-dependent increase in the number of IFN-gamma-secreting T cells in mitogen-activated PBMCs, whereas at higher concentrations the effect was abolished. The proinflammatory effect of statins was not seen in purified T cells per se activated with mitogen. However, conditioned medium derived from statin-treated PBMCs enhanced the number of IFN-gamma-secreting cells in activated purified T cells. This effect was not blocked by mevalonate, but was abolished by neutralizing Abs to IL-18 and IL-12. Similarly, the up-regulation of IFN-gamma-secreting T cells in PBMCs costimulated with statins and mitogens was blocked by the neutralizing anti-IL-18 and anti-IL-12. We showed that simvastatin stimulates the secretion of IL-18 and IL-1beta in monocytes. Active caspase-1, which is required for the processing and secretion of IL-18 and IL-1beta, was activated in simvastatin-treated monocytes. This was blocked by mevalonate and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone. Taken together, the proinflammatory response mediated by statins in activated PBMCs is mediated mainly via the activation of caspase-1 and IL-18 secretion in the monocytes and to a lesser extent by IL-12.

  19. Prickly pear (Opuntia sp.) pectin alters hepatic cholesterol metabolism without affecting cholesterol absorption in guinea pigs fed a hypercholesterolemic diet.

    PubMed

    Fernandez, M L; Lin, E C; Trejo, A; McNamara, D J

    1994-06-01

    Prickly pear pectin intake decreases plasma LDL concentrations by increasing hepatic apolipoprotein B/E receptor expression in guinea pigs fed a hypercholesterolemic diet. To investigate whether prickly pear pectin has an effect on cholesterol absorption and on enzymes responsible for hepatic cholesterol homeostasis, guinea pigs were fed one of three semipurified diets, each containing 15 g lard/100 g diet: 1) the lard-basal diet with no added cholesterol or prickly pear pectin (LB diet); 2) the LB diet with 0.25 g added cholesterol/100 g diet (LC diet); or 3) the LC diet containing 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose (LC-P diet). Animals fed the LB diet had the lowest plasma LDL and hepatic cholesterol concentrations, followed by animals fed the LC-P diet (P < 0.001). Hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was highest in the group fed the LB diet, with similar values for animals in the other two groups. A positive correlation existed between plasma LDL cholesterol concentration and hepatic acyl CoA:cholesterol acyltransferase activity (r = 0.87, P < 0.001). Cholesterol absorption was not different among the three dietary groups. These results indicate that the decreased plasma and hepatic cholesterol concentrations of animals fed prickly pear pectin are not explained by differences in cholesterol absorption but rather are due to mechanisms that alter hepatic cholesterol homeostasis, resulting in lower plasma LDL concentrations.

  20. Potato tuber herbivory increases resistance to aboveground lepidopteran herbivores.

    PubMed

    Kumar, Pavan; Ortiz, Erandi Vargas; Garrido, Etzel; Poveda, Katja; Jander, Georg

    2016-09-01

    Plants mediate interactions between aboveground and belowground herbivores. Although effects of root herbivory on foliar herbivores have been documented in several plant species, interactions between tuber-feeding herbivores and foliar herbivores are rarely investigated. We report that localized tuber damage by Tecia solanivora (Guatemalan tuber moth) larvae reduced aboveground Spodoptera exigua (beet armyworm) and Spodoptera frugiperda (fall armyworm) performance on Solanum tuberosum (potato). Conversely, S. exigua leaf damage had no noticeable effect on belowground T. solanivora performance. Tuber infestation by T. solanivora induced systemic plant defenses and elevated resistance to aboveground herbivores. Lipoxygenase 3 (Lox3), which contributes to the synthesis of plant defense signaling molecules, had higher transcript abundance in T. solanivora-infested leaves and tubers than in equivalent control samples. Foliar expression of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and 3-hydroxy-3-methylglutaryl CoA reductase I (HMGR1) genes, which are involved in chlorogenic acid and steroidal glycoalkaloid biosynthesis, respectively, also increased in response to tuber herbivory. Leaf metabolite profiling demonstrated the accumulation of unknown metabolites as well as the known potato defense compounds chlorogenic acid, α-solanine, and α-chaconine. When added to insect diet at concentrations similar to those found in potato leaves, chlorogenic acid, α-solanine, and α-chaconine all reduced S. exigua larval growth. Thus, despite the fact that tubers are a metabolic sink tissue, T. solanivora feeding elicits a systemic signal that induces aboveground resistance against S. exigua and S. frugiperda by increasing foliar abundance of defensive metabolites.

  1. Targeted disruption of carcinoembryonic antigen-related cell adhesion molecule 1 promotes diet-induced hepatic steatosis and insulin resistance.

    PubMed

    Xu, Elaine; Dubois, Marie-Julie; Leung, Nelly; Charbonneau, Alexandre; Turbide, Claire; Avramoglu, Rita Kohen; DeMarte, Luisa; Elchebly, Mounib; Streichert, Thomas; Lévy, Emile; Beauchemin, Nicole; Marette, André

    2009-08-01

    Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) is a cell adhesion molecule within the Ig superfamily. The Tyr-phosphorylated isoform of CC1 (CC1-L) plays an important metabolic role in the regulation of hepatic insulin clearance. In this report, we show that CC1-deficient (Cc1(-/-)) mice are prone to hepatic steatosis, as revealed by significantly elevated hepatic triglyceride and both total and esterified cholesterol levels compared with age-matched wild-type controls. Cc1(-/-) mice were also predisposed to lipid-induced hepatic steatosis and dysfunction as indicated by their greater susceptibility to store lipids and express elevated levels of enzymatic markers of liver damage after chronic feeding of a high-fat diet. Hepatic steatosis in the Cc1(-/-) mice was linked to a significant increase in the expression of key lipogenic (fatty acid synthase, acetyl CoA carboxylase) and cholesterol synthetic (3-hydroxy-3-methylglutaryl-coenzyme A reductase) enzymes under the control of sterol regulatory element binding proteins-1c and -2 transcription factors. Cc1(-/-) mice also exhibited impaired insulin clearance, glucose intolerance, liver insulin resistance, and elevated hepatic expression of the key gluconeogenic transcriptional activators peroxisome proliferator-activated receptor-gamma coactivator-1 and Forkhead box O1. Lack of CC1 also exacerbated both glucose intolerance and hepatic insulin resistance induced by high-fat feeding, but insulin clearance was not further deteriorated in the high-fat-fed Cc1(-/-) mice. In conclusion, our data indicate that CC1 is a key regulator of hepatic lipogenesis and that Cc1(-/-) mice are predisposed to liver steatosis, leading to hepatic insulin resistance and liver damage, particularly when chronically exposed to dietary fat.

  2. A clinical trial of lovastatin for modification of biomarkers associated with breast cancer risk.

    PubMed

    Vinayak, Shaveta; Schwartz, Erich J; Jensen, Kristin; Lipson, Jafi; Alli, Elizabeth; McPherson, Lisa; Fernandez, Adrian M; Sharma, Vandana B; Staton, Ashley; Mills, Meredith A; Schackmann, Elizabeth A; Telli, Melinda L; Kardashian, Ani; Ford, James M; Kurian, Allison W

    2013-11-01

    Pre-clinical and epidemiologic studies provide rationale for evaluating lipophilic statins for breast cancer prevention. We conducted a single-arm, biomarker modulation trial of lovastatin among women with increased risk of breast cancer. Eligibility criteria included a deleterious germline mutation in BRCA1, BRCA2, CDH1, or TP53; lifetime breast cancer risk of ≥20 % as estimated by the Claus model; or personal history of estrogen receptor and progesterone receptor-negative breast cancer. Participants received 40 mg of lovastatin orally twice daily for 6 months. We evaluated the following biomarkers before and after lovastatin use: breast duct cytology (primary endpoint), serum lipids, C-reactive protein, insulin-like growth factor-1, IGF binding protein-3, lipid peroxidation, oxidative DNA damage, 3-hydroxy-3-methylglutaryl CoA reductase genotype, and mammographic density. Thirty women were enrolled, and 26 (86.7 %) completed the study. For the primary endpoint of changes in breast duct cytology sampled by random periareolar fine needle aspiration, most participants [57.7 %, 95 % confidence interval (CI) 38.9-74.5 %] showed no change after lovastatin; 19.2 % (CI 8.1-38.3 %) had a favorable change in cytology, 7.7 % (95 % CI 1.0-25.3 %) had an unfavorable change, and 15.4 % (95 % CI 5.5-34.2 %) had equivocal results due to acellular specimens, usually after lovastatin. No significant changes were observed in secondary biomarker endpoints. The study was generally well-tolerated: 4 (13.3 %) participants did not complete the study, and one (3.8 %) required a dose reduction. This trial was technically feasible, but demonstrated no significant biomarker modulation; contributing factors may include insufficient sample size, drug dose and/or duration. The results are inconclusive and do not exclude a favorable effect on breast cancer risk.

  3. Metabolic cross-talk between pathways of terpenoid backbone biosynthesis in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Kutzner, Erika; Huber, Claudia; Segura, Juan; Eisenreich, Wolfgang; Arrillaga, Isabel

    2015-10-01

    The metabolic cross-talk between the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in developing spike lavender (Lavandula latifolia Med) was analyzed using specific inhibitors and on the basis of (13)C-labeling experiments. The presence of mevinolin (MEV), an inhibitor of the MVA pathway, at concentrations higher than 0.5 μM significantly reduced plant development, but not the synthesis of chlorophylls and carotenoids. On the other hand, fosmidomycin (FSM), an inhibitor of the MEP pathway, at concentrations higher than 20 μM blocked the synthesis of chlorophyll, carotenoids and essential oils, and significantly reduced stem development. Notably, 1.2 mM MVA could recover the phenotype of MEV-treated plants, including the normal growth and development of roots, and could partially restore the biosynthesis of photosynthetic pigments and, to a lesser extent, of the essential oils in plantlets treated with FSM. Spike lavender shoot apices were also used in (13)C-labeling experiments, where the plantlets were grown in the presence of [U-(13)C6]glucose. GC-MS-analysis of 1,8-cineole and camphor indicated that the C5-precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of both monoterpenes are predominantly biosynthesized via the methylerythritol phosphate (MEP) pathway. However, on the basis of the isotopologue profiles, a minor contribution of the MVA pathway was evident that was increased in transgenic spike lavender plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the first enzyme of the MVA pathway. Together, these findings provide evidence for a transport of MVA-derived precursors from the cytosol to the plastids in leaves of spike lavender.

  4. Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

    PubMed Central

    Rodríguez-Vita, Juan; Sánchez-Galán, Eva; Santamaría, Beatriz; Sánchez-López, Elsa; Rodrigues-Díez, Raquel; Blanco-Colio, Luís Miguel; Egido, Jesús; Ortiz, Alberto; Ruiz-Ortega, Marta

    2008-01-01

    Background The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells. Methodology In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected. Conclusions Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs. PMID:19088845

  5. Maternal dietary betaine supplementation modifies hepatic expression of cholesterol metabolic genes via epigenetic mechanisms in newborn piglets.

    PubMed

    Cai, Demin; Jia, Yimin; Lu, Jingyu; Yuan, Mengjie; Sui, Shiyan; Song, Haogang; Zhao, Ruqian

    2014-11-14

    To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory element-binding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27α-hydroxylase (CYP27α1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7α-hydroxylase (CYP7α1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.

  6. Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.

    PubMed

    Mo, H; Elson, C E

    1999-04-01

    Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable

  7. Cardiovascular effects of statins, beyond lipid-lowering properties.

    PubMed

    Mihos, Christos G; Pineda, Andres M; Santana, Orlando

    2014-10-01

    The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, better known as 'statins', are amongst the most widely used medications in the world. They have become a pivotal component in the primary and secondary prevention of coronary artery and vascular disease. However, a growing amount of evidence has suggested that statins also possess strong pleiotropic effects irrespective of their lipid-lowering properties, which include enhancement of endothelial function, anti-inflammatory and anti-atherothrombotic properties, and immunomodulation. The following provides a comprehensive and updated review of the clinical evidence regarding the pleiotropic effects of statins in cardiovascular disorders and their potential therapeutic benefits.

  8. Statin-induced necrotizing myositis - a discrete autoimmune entity within the "statin-induced myopathy spectrum".

    PubMed

    Hamann, Philip D H; Cooper, Robert G; McHugh, Neil J; Chinoy, Hector

    2013-10-01

    Statin-induced necrotizing myositis is increasingly being recognised as part of the "statin-induced myopathy spectrum". As in other immune-mediated necrotizing myopathies, statin-induced myositis is characterised by proximal muscle weakness with marked serum creatinine kinase elevations and histological evidence of myonecrosis, with little or no inflammatory cell infiltration. Unlike other necrotizing myopathies, statin-induced myopathy is associated with the presence of autoantibodies directed against 3-hydroxy-3-methylglutaryl- coenzyme A reductase (the enzyme target of statin therapies), and with Human Leukocyte Antigen-DRB1*11. This article summarises the clinical presentation, investigations and management of this rare, but serious complication of statin therapy.

  9. Thioredoxin reductase.

    PubMed Central

    Mustacich, D; Powis, G

    2000-01-01

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  10. Global Hawk Pacific (GloPac) COA and Mission Coordination

    NASA Technical Reports Server (NTRS)

    Dillon, Mark; Hall, Philip

    2010-01-01

    This slide presentation reviews the science objectives of the Global Hawk unmanned aircraft system (UAS) in the Pacific region, shows examp le flight tracks, the satellite under-flight requirement, the flight planning, and the agencies coordination of the airspace required for the Certificate of Authorization (COA).

  11. Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA.

    PubMed

    Uchino, Keiichi; Saito, Terumi; Gebauer, Birgit; Jendrossek, Dieter

    2007-11-01

    Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from (14)C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD(+), indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD(+)/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

  12. Effects of excess dietary tyrosine or certain xenobiotics on the cholesterogenesis in rats

    SciTech Connect

    Nagaoka, S.; Masaki, H.; Aoyama, Y.; Yoshida, A.

    1986-05-01

    Comparison of the effects of excess dietary tyrosine, DDT, chlorobutanol (Chloretone) or butylated hydroxyanisole (BHA) on serum cholesterol, hepatic activities of the rate-limiting enzyme of cholesterol synthesis,3-hydroxy-3-methylglutaryl coenzyme A reductase and in vivo rates of the hepatic cholesterol synthesis measured by /sup 3/H/sub 2/O incorporation were investigated in rats. Serum cholesterol concentration was significantly higher in rats fed the DDT, chlorobutanol, BHA or excess tyrosine diets than in rats fed the control diet for 7 days. Serum cholesterol concentration remained higher compared to control rats when excess tyrosine was fed for 21 d. When rats were fed a basal diet after feeding a tyrosine excess diet for 2 wk, liver weight and serum cholesterol level returned to normal within 7 d. The incorporation of /sup 3/H/sub 2/O into liver cholesterol and the activity of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase were greater in rats fed excess tyrosine or certain xenobiotics than in control rats. Present results suggested that the increase in serum cholesterol concentration due to excess dietary tyrosine or certain xenobiotics is mainly attributable to the stimulation of liver cholesterol synthesis.

  13. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    NASA Astrophysics Data System (ADS)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 °C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 °C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  14. Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

    NASA Astrophysics Data System (ADS)

    Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

    In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

  15. Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death.

    PubMed

    Pandyra, Aleksandra A; Mullen, Peter J; Goard, Carolyn A; Ericson, Elke; Sharma, Piyush; Kalkat, Manpreet; Yu, Rosemary; Pong, Janice T; Brown, Kevin R; Hart, Traver; Gebbia, Marinella; Lang, Karl S; Giaever, Guri; Nislow, Corey; Moffat, Jason; Penn, Linda Z

    2015-09-29

    The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy.

  16. Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death

    PubMed Central

    Pandyra, Aleksandra A.; Mullen, Peter J.; Goard, Carolyn A.; Ericson, Elke; Sharma, Piyush; Kalkat, Manpreet; Yu, Rosemary; Pong, Janice T.; Brown, Kevin R.; Hart, Traver; Gebbia, Marinella; Lang, Karl S.; Giaever, Guri; Nislow, Corey; Moffat, Jason; Penn, Linda Z.

    2015-01-01

    The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy. PMID:26353928

  17. Transcriptome and gene expression analysis in cold-acclimated guayule (Parthenium argentatum) rubber-producing tissue.

    PubMed

    Ponciano, Grisel; McMahan, Colleen M; Xie, Wenshuang; Lazo, Gerard R; Coffelt, Terry A; Collins-Silva, Jillian; Nural-Taban, Aise; Gollery, Martin; Shintani, David K; Whalen, Maureen C

    2012-07-01

    Natural rubber biosynthesis in guayule (Parthenium argentatum Gray) is associated with moderately cold night temperatures. To begin to dissect the molecular events triggered by cold temperatures that govern rubber synthesis induction in guayule, the transcriptome of bark tissue, where rubber is produced, was investigated. A total of 11,748 quality expressed sequence tags (ESTs) were obtained. The vast majority of ESTs encoded proteins that are similar to stress-related proteins, whereas those encoding rubber biosynthesis-related proteins comprised just over one percent of the ESTs. Sequence information derived from the ESTs was used to design primers for quantitative analysis of the expression of genes that encode selected enzymes and proteins with potential impact on rubber biosynthesis in field-grown guayule plants, including 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, small rubber particle protein, allene oxide synthase, and cis-prenyl transferase. Gene expression was studied for field-grown plants during the normal course of seasonal variation in temperature (monthly average maximum 41.7 °C to minimum 0 °C, from November 2005 through March 2007) and rubber transferase enzymatic activity was also evaluated. Levels of gene expression did not correlate with air temperatures nor with rubber transferase activity. Interestingly, a sudden increase in night temperature 10 days before harvest took place in advance of the highest CPT gene expression level.

  18. Volatiles Emitted at Different Flowering Stages of Jasminum sambac and Expression of Genes Related to α-Farnesene Biosynthesis.

    PubMed

    Yu, Ying; Lyu, Shiheng; Chen, Dan; Lin, Yi; Chen, Jianjun; Chen, Guixin; Ye, Naixing

    2017-03-29

    Fresh jasmine flowers have been used to make jasmine teas in China, but there has been no complete information about volatile organic compound emissions in relation to flower developmental stages and no science-based knowledge about which floral stage should be used for the infusion. This study monitored volatile organic compounds emitted from living flowers of Jasminum sambac (L.) Ait. 'Bifoliatum' at five developmental stages and also from excised flowers. Among the compounds identified, α-farnesene, linalool, and benzyl acetate were most abundant. Since α-farnesene is synthesized through the Mevalonate pathway, four genes encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase, and terpene synthase were isolated. Their expression patterns in living flowers at the five stages and in excised flowers coincided with the emission patterns of α-farnesene. Application of lovastatin, a HMGR inhibitor, significantly reduced the expression of the genes and greatly decreased the emission of α-farnesene. The sweet scent was diminished from lovastatin-treated flowers as well. These results indicate that α-farnesene is an important compound emitted from jasmine flowers, and its emission patterns suggest that flowers at the opening stage or flower buds 8 h after excision should be used for the infusion of tea leaves.

  19. Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases.

    PubMed

    Eberlein, Christian; Estelmann, Sebastian; Seifert, Jana; von Bergen, Martin; Müller, Michael; Meckenstock, Rainer U; Boll, Matthias

    2013-06-01

    The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

  20. Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes.

    PubMed

    Fujioka, T; Tsujita, Y; Shimotsu, H

    1997-06-11

    We examined the effect of pravastatin sodium (pravastatin), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on fatty acid synthesis in rat liver. The repeated administration of pravastatin to rats at 250 mg/kg for 7 days led to a 2.8-fold increase in fatty acid synthesis in the liver. The diurnal change of fatty acid synthesis was not affected by the treatment. Hepatic fatty acid synthase activity was increased 3.2-fold, while acetyl-CoA carboxylase activity was not changed by the repeated administration of pravastatin. In rat hepatocytes, the incubation with 2 microg/ml pravastatin for 24 h increased fatty acid synthase activity 1.5-fold, as well as HMG-CoA reductase activity 2.8-fold. These results suggest that HMG-CoA reductase inhibitors might increase fatty acid synthesis in vivo through the induction of hepatic fatty acid synthase.

  1. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.

  2. Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria

    PubMed Central

    Puisac, Beatriz; Arnedo, María; Casale, Cesar H.; Ribate, María Pilar; Castiella, Tomás; Ramos, Feliciano J.; Ribes, Antonia; Pérez-Cerdá, Celia; Casals, Nuria; Hegardt, Fausto G.

    2010-01-01

    3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients. PMID:20532825

  3. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  4. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    SciTech Connect

    Roman-Lopez, C.R.; Allred, J.B.

    1986-05-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind (/sup 14/C)methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase.

  5. Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis

    PubMed Central

    Ghosh, Alok; Pratt, Anthony T.; Soma, Shivatheja; Theriault, Sarah G.; Griffin, Aaron T.; Trivedi, Prachi P.; Gohil, Vishal M.

    2016-01-01

    Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues the coa6Δ phenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins. Furthermore, we show that patient mutations in Coa6 disrupt Coa6–Cox2 interaction, providing the biochemical basis for disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis. PMID:26669719

  6. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    PubMed

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

  7. Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

    PubMed

    Baertling, Fabian; A M van den Brand, Mariel; Hertecant, Jozef L; Al-Shamsi, Aisha; P van den Heuvel, Lambert; Distelmaier, Felix; Mayatepek, Ertan; Smeitink, Jan A; Nijtmans, Leo G J; Rodenburg, Richard J T

    2015-01-01

    COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.

  8. Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases.

    PubMed

    Zhang, Wei; Shrestha, Ruben; Buckley, Rachael M; Jewell, Jamie; Bossmann, Stefan H; Stubbe, JoAnne; Li, Ping

    2014-08-15

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

  9. Comparative evaluation of HMG CoA reductase inhibitors in experimentally-induced myocardial necrosis: Biochemical, morphological and histological studies.

    PubMed

    Variya, Bhavesh C; Patel, Snehal S; Trivedi, Jinal I; Gandhi, Hardik P; Rathod, S P

    2015-10-05

    The present study was carried out to evaluate the protective effect of different statins on isoproterenol (ISO) induced myocardial necrosis. Atorvastatin, rosuvastatin, fluvastatin, simvastatin and pravastatin (10 mg/kg/day) were administered for 12 weeks. After pretreatment of 12 weeks myocardial necrosis was induced by subsequent injection of ISO (85 mg/kg/day, s.c.) to wistar rats. Serum biochemical parameters like glucose, lipid profile, cardiac markers and transaminases were evaluated. Animals were killed and heart was excised for histopathology and antioxidant study. Statins pretreated rats showed significant protection against ISO induced elevation in serum biochemical parameters and serum level of cardiac marker enzymes and transaminase level as compared to ISO control group. Mild to moderate protection was observed in different statins treated heart in histopathology and TTC stained sections. Result from our study also revealed that statins could efficiently protect against ISO intoxicated myocardial necrosis by impairing membrane bound enzyme integrity and endogenous antioxidant enzyme levels. Amongst all statins used, rosuvastatin and pravastatin were found to have maximum cardio-protective activity against ISO induced myocardial necrosis as compared to other statins.

  10. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  11. Coenzyme Q10 and statin-related myopathy.

    PubMed

    2015-05-01

    Statins inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which is involved in the production of mevalonic acid in the cholesterol biosynthesis pathway. This pathway also results in the production of other bioactive molecules including coenzyme Q10 (also known as ubiquinone or ubidecarenone). Coenzyme Q10 is a naturally-occurring coenzyme with antioxidant effects that is involved in electron transport in mitochondria and is thought to play a role in energy transfer in skeletal muscle. Muscle-related problems are a frequently reported adverse effect of statins, and it has been hypothesised that a reduced endogenous coenzyme Q10 concentration is a cause of statin-induced myopathy. Coenzyme Q10 supplementation has therefore been proposed to reduce the adverse muscular effects sometimes seen with statins. Here, we consider whether coenzyme Q10 has a place in the management of statin-induced myopathy.

  12. Molecular mechanisms underlying the effects of statins in the central nervous system.

    PubMed

    McFarland, Amelia J; Anoopkumar-Dukie, Shailendra; Arora, Devinder S; Grant, Gary D; McDermott, Catherine M; Perkins, Anthony V; Davey, Andrew K

    2014-11-10

    3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly referred to as statins, are widely used in the treatment of dyslipidaemia, in addition to providing primary and secondary prevention against cardiovascular disease and stroke. Statins' effects on the central nervous system (CNS), particularly on cognition and neurological disorders such as stroke and multiple sclerosis, have received increasing attention in recent years, both within the scientific community and in the media. Current understanding of statins' effects is limited by a lack of mechanism-based studies, as well as the assumption that all statins have the same pharmacological effect in the central nervous system. This review aims to provide an updated discussion on the molecular mechanisms contributing to statins' possible effects on cognitive function, neurodegenerative disease, and various neurological disorders such as stroke, epilepsy, depression and CNS cancers. Additionally, the pharmacokinetic differences between statins and how these may result in statin-specific neurological effects are also discussed.

  13. Statins in oncological research: from experimental studies to clinical practice.

    PubMed

    Kubatka, Peter; Kruzliak, Peter; Rotrekl, Vladimir; Jelinkova, Sarka; Mladosievicova, Beata

    2014-12-01

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are commonly used drugs in the treatment of dyslipidemias, primarily raised cholesterol. Recently, many epidemiological and preclinical studies pointed to anti-tumor properties of statins, including anti-proliferative activities, apoptosis, decreased angiogenesis and metastasis. These processes play an important role in carcinogenesis and, therefore, the role of statins in cancer disease is being seriously discussed among oncologists. Anti-neoplastic properties of statins combined with an acceptable toxicity profile in the majority of individuals support their further development as anti-tumor drugs. The mechanism of action, current preclinical studies and clinical efficacy of statins are reviewed in this paper. Moreover, promising results have been reported regarding the statins' efficacy in some cancer types, especially in esophageal and colorectal cancers, and hepatocellular carcinoma. Statins' hepatotoxicity has traditionally represented an obstacle to the prescription of this class of drugs and this issue is also discussed in this review.

  14. Pleiotropic vascular protective effects of statins in perioperative medicine.

    PubMed

    Fang, Shin-Yuan; Roan, Jun-Neng; Luo, Chwan-Yau; Tsai, Yu-Chuan; Lam, Chen-Fuh

    2013-09-01

    3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (statins) is one of the most commonly prescribed agents for controlling hyperlipidemia. Apart from their lipid-lowering property, statins are well known for their pleiotropic effects, such as improvement of vascular endothelial dysfunction, attenuation of inflammatory responses, stabilization of atherosclerotic plaques, inhibition of vascular smooth muscle proliferation, and modulation of procoagulant activity and platelet function. The vasculo-protective effect of statins is mainly mediated by inhibition of the mevalonate pathway and oxidized low-density lipoprotein generation, thereby enhancing the biosynthesis of endothelium-derived nitric oxide. Accumulating clinical evidence strongly suggests that administration of statins reduces overall mortality, the development myocardial infarction and atrial fibrillation, and length of hospital stay after a major cardiac/noncardiac surgery. This review updates the clinical pharmacology and therapeutic applications of statins during major operations, and highlights the anesthesia considerations for perioperative statin therapy.

  15. Reduced mitochondrial coenzyme Q10 levels in HepG2 cells treated with high-dose simvastatin: A possible role in statin-induced hepatotoxicity?

    SciTech Connect

    Tavintharan, S. Ong, C.N.; Jeyaseelan, K.; Sivakumar, M.; Lim, S.C.; Sum, C.F.

    2007-09-01

    Lowering of low-density lipoprotein cholesterol is well achieved by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins). Statins inhibit the conversion of HMG-CoA to mevalonate, a precursor for cholesterol and coenzyme Q10 (CoQ{sub 10}). In HepG2 cells, simvastatin decreased mitochondrial CoQ{sub 10} levels, and at higher concentrations was associated with a moderately higher degree of cell death, increased DNA oxidative damage and a reduction in ATP synthesis. Supplementation of CoQ{sub 10}, reduced cell death and DNA oxidative stress, and increased ATP synthesis. It is suggested that CoQ{sub 10} deficiency plays an important role in statin-induced hepatopathy, and that CoQ{sub 10} supplementation protects HepG2 cells from this complication.

  16. Effect of Selenium-Enriched Agaricus bisporus (Higher Basidiomycetes) Extracts, Obtained by Pressurized Water Extraction, on the Expression of Cholesterol Homeostasis Related Genes by Low-Density Array.

    PubMed

    Gil-Ramírez, Alicia; Soler-Rivas, Cristina; Rodriguez-Casado, Arantxa; Ruiz-Rodríguez, Alejandro; Reglero, Guillermo; Marín, Francisco Ramón

    2015-01-01

    Culinary-medicinal mushrooms are able to lower blood cholesterol levels in animal models by different mechanisms. They might impair the endogenous cholesterol synthesis and exogenous cholesterol absorption during digestion. Mushroom extracts, obtained using pressurized water extractions (PWE) from Agaricus bisporus basidiomes, supplemented or not supplemented with selenium, were applied to HepG2 cell cultures to study the expression of 19 genes related to cholesterol homeostasis by low-density arrays (LDA). Only the PWE fractions obtained at 25°C showed 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitory activity. Besides the enzymatic inhibition, PWE extracts may downregulate some of the key genes involved in the cholesterol homeostasis, such as the squalene synthase gene (FDFT1), since its mRNA expression falls by one third of its initial value. In summary, A. bisporus extracts may also modulate biological cholesterol levels by molecular mechanisms further than the enzymatic way previously reported.

  17. An ABC transporter controls export of a Drosophila germ cell attractant.

    PubMed

    Ricardo, Sara; Lehmann, Ruth

    2009-02-13

    Directed cell migration, which is critical for embryonic development, leukocyte trafficking, and cell metastasis, depends on chemoattraction. 3-hydroxy-3-methylglutaryl coenzyme A reductase regulates the production of an attractant for Drosophila germ cells that may itself be geranylated. Chemoattractants are commonly secreted through a classical, signal peptide-dependent pathway, but a geranyl-modified attractant would require an alternative pathway. In budding yeast, pheromones produced by a-cells are farnesylated and secreted in a signal peptide-independent manner, requiring the adenosine triphosphate-binding cassette (ABC) transporter Ste6p. Here we show that Drosophila germ cell migration uses a similar pathway, demonstrating that invertebrate germ cells, like yeast cells, are attracted to lipid-modified peptides. Components of this unconventional export pathway are highly conserved, suggesting that this pathway may control the production of similarly modified chemoattractants in organisms ranging from yeast to humans.

  18. Statin-associated myopathy and its exacerbation with exercise.

    PubMed

    Meador, Benjamin M; Huey, Kimberly A

    2010-10-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are a common and effective treatment for hypercholesterolemia, with a low overall rate of side-effects. The most common complication is some degree of skeletal muscle myopathy, ranging from painless serum creatine kinase elevations to rhabdomyolysis. Unfortunately, the likelihood and/or severity of complications increases with the combination of statin treatment and physical activity. The specific pathways that mediate statin-associated myopathy are unclear, and research directly addressing the exacerbation with exercise is limited. Potential mechanisms include the induction of skeletal muscle fiber apoptosis, alterations in ubiquitin-proteasome pathway activity, mitochondrial dysfunction, and terpenoid depletion. In this review we provide an overview of research that specifically addresses the combination of statin-associated myopathy and physical activity and highlight some deficiencies in the available literature, as well as future directions for this important subset of statin-associated myopathy.

  19. Statin-associated autoimmune myopathy and anti-HMGCR autoantibodies.

    PubMed

    Mohassel, Payam; Mammen, Andrew L

    2013-10-01

    Statins are among the most commonly prescribed medications that significantly reduce cardiovascular risk in selected individuals. However, these drugs can also be associated with muscle symptoms ranging from mild myalgias to severe rhabdomyolysis. Although statin myotoxicity is usually self-limited, in some instances statin-exposed subjects can develop an autoimmune myopathy typically characterized by progressive weakness, muscle enzyme elevations, a necrotizing myopathy on muscle biopsy, and autoantibodies that recognize 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the pharmacologic target of statins. These antibodies are also found in some autoimmune myopathy patients without statin exposure. Importantly, anti-HMGCR antibodies are not found in the vast majority of statin-exposed subjects without autoimmune myopathy, including those with self-limited statin intolerance. Thus, testing for these antibodies may help differentiate those with self-limited statin myopathy who recover after statin discontinuation from those with a progressive statin-associated autoimmune myopathy who typically require immunosuppressive therapy.

  20. Mechanisms and assessment of statin-related muscular adverse effects.

    PubMed

    Moßhammer, Dirk; Schaeffeler, Elke; Schwab, Matthias; Mörike, Klaus

    2014-09-01

    Statin-associated muscular adverse effects cover a wide range of symptoms, including asymptomatic increase of creatine kinase serum activity and life-threatening rhabdomyolysis. Different underlying pathomechanisms have been proposed. However, a unifying concept of the pathogenesis of statin-related muscular adverse effects has not emerged so far. In this review, we attempt to categorize these mechanisms along three levels. Firstly, among pharmacokinetic factors, it has been shown for some statins that inhibition of cytochrome P450-mediated hepatic biotransformation and hepatic uptake by transporter proteins contribute to an increase of systemic statin concentrations. Secondly, at the myocyte membrane level, cell membrane uptake transporters affect intracellular statin concentrations. Thirdly, at the intracellular level, inhibition of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase results in decreased intracellular concentrations of downstream metabolites (e.g. selenoproteins, ubiquinone, cholesterol) and alteration of gene expression (e.g. ryanodine receptor 3, glycine amidinotransferase). We also review current recommendations for prescribers.

  1. Place of pitavastatin in the statin armamentarium: promising evidence for a role in diabetes mellitus.

    PubMed

    Kawai, Yasuyuki; Sato-Ishida, Ryoko; Motoyama, Atsushi; Kajinami, Kouji

    2011-01-01

    Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, known as statins, have revolutionized the treatment of hypercholesterolemia and coronary artery disease prevention. However, there are considerable issues regarding statin safety and further development of residual risk control, particularly for diabetic and metabolic syndrome patients. Pitavastatin is a potent statin with low-density lipoprotein (LDL) cholesterol-lowering effects comparable to those of atorvastatin or rosuvastatin. Pitavastatin has a high-density lipoprotein (HDL) cholesterol raising effect, may improve insulin resistance, and has little influence on glucose metabolism. Considering these factors along with its unique pharmacokinetic properties, which suggest minimal drug-drug interaction, pitavastatin could provide an alternative treatment choice, especially in patients with glucose intolerance or diabetes mellitus. Many clinical trials are now underway to test the clinical efficacy of pitavastatin in various settings and are expected to provide further information.

  2. Quantification of the N-desmethyl metabolite of rosuvastatin in human plasma by automated SPE followed by HPLC with tandem MS detection.

    PubMed

    Hull, Caroline K; Martin, Paul D; Warwick, Michael J; Thomas, Elizabeth

    2004-05-28

    A selective, accurate and precise assay was developed for the quantification in human plasma of the N-desmethyl metabolite of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor rosuvastatin. The assay-employing automated SPE followed by HPLC with positive ion electrospray tandem MS (HPLC-MS/MS)-was validated. The standard curve range for N-desmethyl rosuvastatin in human plasma was 0.5-30 ng/ml with 0.5 ng/ml being the limit of quantification. Plasma samples were mixed 1:1 with sodium acetate buffer (pH 4.0; 0.1M) soon after separation from red blood cells. N-Desmethyl rosuvastatin was stable in plasma:buffer at room temperature for 24h and at -70 degrees C for 12 months. The assay was applied successfully to the quantification of N-desmethyl rosuvastatin in human plasma following administration of rosuvastatin.

  3. Statin-associated rhabdomyolysis with acute renal failure complicated by intradialytic NSTEMI: a review of lipid management considerations.

    PubMed

    Kar, Subrata; Chockalingam, Anand

    2013-01-01

    Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are associated with myopathy, myalgias, myositis, and rhabdomyolysis. Rhabdoymyolysis is a rare complication and may cause acute renal failure, which may be fatal. In such cases, alternative therapies should be considered. In this review, we attempted to elucidate the lipid management options in patients with rhabdomyolysis and coronary artery disease. We also describe a case report of a patient who developed rhabdomyolysis from dual antilipid therapy followed by acute renal failure and non-ST elevation myocardial infarction. Such a complex case has not been reported in the literature, and lipid management options may include niacin, omega 3-fatty acids, or bile acid sequestrants. Once alternative therapies are initiated, monitoring a patient closely with evaluation for associated adverse events should be performed.

  4. Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males.

    PubMed

    Prchalová, Darina; Buček, Aleš; Brabcová, Jana; Žáček, Petr; Kindl, Jiří; Valterová, Irena; Pichová, Iva

    2016-02-02

    Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males.

  5. A novel therapeutic effect of statins on nephrogenic diabetes insipidus.

    PubMed

    Bonfrate, Leonilde; Procino, Giuseppe; Wang, David Q-H; Svelto, Maria; Portincasa, Piero

    2015-02-01

    Statins competitively inhibit hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase, resulting in reduced plasma total and low-density lipoprotein cholesterol levels. Recently, it has been shown that statins exert additional 'pleiotropic' effects by increasing expression levels of the membrane water channels aquaporin 2 (AQP2). AQP2 is localized mainly in the kidney and plays a critical role in determining cellular water content. This additional effect is independent of cholesterol homoeostasis, and depends on depletion of mevalonate-derived intermediates of sterol synthetic pathways, i.e. farnesylpyrophosphate and geranylgeranylpyrophosphate. By up-regulating the expression levels of AQP2, statins increase water reabsorption by the kidney, thus opening up a new avenue in treating patients with nephrogenic diabetes insipidus (NDI), a hereditary disease that yet lacks high-powered and limited side effects therapy. Aspects related to water balance determined by AQP2 in the kidney, as well as standard and novel therapeutic strategies of NDI are discussed.

  6. Lovastatin-induced RhoA modulation and its effect on senescence in prostate cancer cells

    SciTech Connect

    Lee, Jeeyun; Lee, Inkyoung; Park, Chaehwa; Kang, Won Ki . E-mail: wkkang@smc.samsung.co.kr

    2006-01-20

    Lovastatin inhibits a 3-hydroxy 3-methylglutaryl coenzyme A reductase and prevents the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible for important cell signaling in cell proliferation and migration. Recently, the anti-cancer effect of lovastatin has been suggested in various tumor types. In this study, we showed that a low dose lovastatin induced senescence and G1 cell cycle arrest in human prostate cancer cells. Addition of GGPP or mevalonate, but not FPP, prevented the lovastatin-induced G1 phase cell cycle arrest and cell senescence. We found that constitutively active RhoA (caRhoA) reversed lovastatin-induced senescence in caRhoA-transfected PC-3 cells. Thus, we postulate that modulation of RhoA may be critical in lovastatin-induced senescence in PC-3 cells.

  7. Lipid-altering therapy and atrial fibrillation.

    PubMed

    Bachmann, Justin M; Majmudar, Maulik; Tompkins, Christine; Blumenthal, Roger S; Marine, Joseph E

    2008-01-01

    Atrial fibrillation (AF) is a common cardiac arrhythmia with significant morbidity and public health cost. Because of limitations of efficacy and safety of conventional antiarrhythmic agents, alternative therapies for AF are needed. The potential antiarrhythmic properties of lipid-altering therapy, including the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and fish oils, are increasingly recognized, particularly in light of their potential anti-inflammatory properties. This review examines the known effects of lipid-altering therapy on atrial arrhythmias in both experimental and clinical settings. Inflammatory states, such as post-cardiac surgery and AF of recent onset, show promise as targets. In contrast, lipid-lowering therapy is less likely to affect longstanding persistent AF. Current recommendations for the use of lipid-altering therapy for prevention and treatment of AF are summarized.

  8. Inhibition of cholesteryl ester formation in human fibroblasts by an analogue of 7-ketocholesterol and by progesterone

    PubMed Central

    Goldstein, Joseph L.; Faust, Jerry R.; Dygos, John H.; Chorvat, Robert J.; Brown, Michaels S.

    1978-01-01

    The synthesis of cholesteryl esters in cultured human fibroblasts is catalyzed by a microsomal acyl-coenzyme A:cholesterol acyltransferase (EC 2.3.1.26). The acyltransferase activity is enhanced when fibroblasts take up cholesterol contained in plasma low density lipoprotein. In the current studies two steroids, SC-31769 (an analogue of 7-ketocholesterol) and progesterone, were shown to inhibit acyltransferase activity in cell-free extracts of human fibroblasts. When added to intact cells, these steroids inhibited the incorporation of [14C]oleate into cellular cholesteryl [14C]oleate and reduced the accumulation of cholesteryl esters in fibroblasts exposed to low density lipoprotein. The inhibition of cholesteryl ester formation in intact cells by SC-31769 and progesterone was readily reversible. Neither compound inhibited the incorporation of [14C]oleate into [14C]triglycerides or [14C]phospholipids. When incubated with fibroblast monolayers at a concentration of 1 μg/ml, SC-31769 suppressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34], the rate-controlling enzyme in cholesterol synthesis. In contrast, progesterone had no effect on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity at concentrations as high as 25 μg/ml. The availability of two types of steroid compounds that inhibit the acyltransferase activity and cholesteryl ester synthesis in human fibroblasts should prove useful in further studies of the regulatory mechanisms responsible for cholesteryl ester accumulation in human cells under normal and pathologic conditions. PMID:205874

  9. Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines.

    PubMed

    Park, Youngki; Carr, Timothy P

    2013-02-01

    Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.

  10. Anti-HMGCR antibodies as a biomarker for immune-mediated necrotizing myopathies: A history of statins and experience from a large international multi-center study.

    PubMed

    Musset, Lucile; Allenbach, Yves; Benveniste, Olivier; Boyer, Olivier; Bossuyt, Xavier; Bentow, Chelsea; Phillips, Joe; Mammen, Andrew; Van Damme, Philip; Westhovens, René; Ghirardello, Anna; Doria, Andrea; Choi, May Y; Fritzler, Marvin J; Schmeling, Heinrike; Muro, Yoshinao; García-De La Torre, Ignacio; Ortiz-Villalvazo, Miguel A; Bizzaro, Nicola; Infantino, Maria; Imbastaro, Tiziana; Peng, Qinglin; Wang, Guochun; Vencovský, Jiří; Klein, Martin; Krystufkova, Olga; Franceschini, Franco; Fredi, Micaela; Hue, Sophie; Belmondo, Thibaut; Danko, Katalin; Mahler, Michael

    2016-10-01

    In an effort to find naturally occurring substances that reduce cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), statins were first discovered by Endo in 1972. With the widespread prescription and use of statins to decrease morbidity from myocardial infarction and stroke, it was noted that approximately 5% of all statin users experienced muscle pain and weakness during treatment. In a smaller proportion of patients, the myopathy progressed to severe morbidity marked by proximal weakness and severe muscle wasting. Remarkably, Mammen and colleagues were the first to discover that the molecular target of statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is an autoantibody target in patients that develop an immune-mediated necrotizing myopathy (IMNM). These observations have been confirmed in a number of studies but, until today, a multi-center, international study of IMNM, related idiopathic inflammatory myopathies (IIM), other auto-inflammatory conditions and controls has not been published. Accordingly, an international, multi-center study investigated the utility of anti-HMGCR antibodies in the diagnosis of statin-associated IMNM in comparison to different forms of IIM and controls. This study included samples from patients with different forms of IIM (n=1250) and patients with other diseases (n=656) that were collected from twelve sites and tested for anti-HMGCR antibodies by ELISA. This study confirmed that anti-HMGCR autoantibodies, when found in conjunction with statin use, characterize a subset of IIM who are older and have necrosis on muscle biopsy. Taken together, the data to date indicates that testing for anti-HMGCR antibodies is important in the differential diagnosis of IIM and might be considered for future classification criteria.

  11. Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

    PubMed

    Faas, F H; Carter, W J; Wynn, J O

    1978-11-22

    The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.

  12. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

    PubMed

    Sattler, Steven A; Walker, Alexander M; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

    2017-02-01

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

  13. Spectroscopic Classification of SN 2017coa as a Type Ia Supernova

    NASA Astrophysics Data System (ADS)

    Xiang, Danfeng; Rui, Liming; Wang, Xiaofeng; Tan, Hanjie; Li, Wenxiong; Zhang, Tianmeng; Xu, Zhijian; Yang, Zesheng; Song, Hao; Mo, Jun; Wang, Yuanhao; Zhou, Ziheng; Meng, Xianmin; Qian, Shenban; Jia, Junjun; Zhou, Xu; Zhang, Jujia

    2017-04-01

    We obtained an optical spectrum (range 360-840 nm) of SN 2017coa,discovered by Tsinghua-NAOC Transient Survey (TNTS), on UT Mar.31.49 2017 with the 2.16-m telescope (+BFOSC) at Xinglong Station of National Astronomical Observatories of China (NAOC).

  14. Response of the cholesterol metabolism to a negative energy balance in dairy cows depends on the lactational stage.

    PubMed

    Gross, Josef J; Kessler, Evelyne C; Albrecht, Christiane; Bruckmaier, Rupert M

    2015-01-01

    The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.

  15. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  16. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  17. Artemisinic acid inhibits melanogenesis through downregulation of C/EBP α-dependent expression of HMG-CoA reductase gene.

    PubMed

    Lee, Jongsung; Lee, Jienny; Jung, Eunsun; Cho, Jae Youl; Park, Deokhoon

    2013-01-01

    Cholesterol is associated with the regulation of melanogenesis which is the major physiological defense against solar irradiation. The present study was designed to determine the effects of artemisinic acid on melanogenesis and its mechanisms of action in human epidermal melanocytes. In this study, we found that artemisinic acid inhibited melanin content. The mRNA levels of microphthalmia-associated transcription factor (MITF) and its downstream genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 were reduced by artemisinic acid treatment. Additionally, the mRNA levels of melanogenesis-related genes (c-KIT, stem cell factor (SCF), and macrophage migration inhibitory factor (MIF)) were down-regulated by artemisinic acid. Furthermore, cAMP production and protein kinase A (PKA) activity were suppressed by artemisinic acid. Moreover, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that artemisinic acid regulated melanogenesis by inhibiting cholesterol synthesis through downregulation of the hydroxymethylglutaryl CoA (HMG CoA) reductase gene, which was mediated through reduced expression of the CCAAT/enhancer-binding protein (C/EBP) α gene. Taken together, these findings indicate that the inhibition of melanogenesis by artemisinic acid occurs through reduced expression of the HMG CoA reductase gene, which is mediated by C/EBP α inhibition and suggest that artemisinic acid may be useful as a hyperpigmentation inhibitor.

  18. Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum.

    PubMed

    Koetsier, Martijn J; Jekel, Peter A; Wijma, Hein J; Bovenberg, Roel A L; Janssen, Dick B

    2011-03-23

    Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids L-phenylalanine and L-tyrosine, as well as the non-proteinogenic amino acids D-phenylalanine, D-tyrosine and (R)- and (S)-β-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-β-phenylalanine, forming (R)-β-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with β-phenylalanines and D-α-phenylalanine.

  19. Mosaic Conservation Opportunity Areas - Liberal Model (ECO_RES.COA_MOSAIC33)

    EPA Pesticide Factsheets

    The COA_Mosaic33 layer designates areas with potential for forest/grassland mosaic conservation. These are areas of natural or semi-natural forest/grassland mosaic land cover patches that are at least 75 meters away from roads and away from patch edges. OAs were modeled by creating distance grids using the National Land Cover Database and the Census Bureau's TIGER roads files

  20. Mosaic Conservation Opportunity Areas - Conservativel Model (ECO_RES.COA_MOSAIC66)

    EPA Pesticide Factsheets

    The COA_Mosaic66 layer designates areas with potential for forest/grassland mosaic conservation. These are areas of natural or semi-natural forest/grassland land cover patches that area at least 395 meters away from roads and away from patch edges. OAs were modeled by creating distance grids using the National Land Cover Database and the Census Bureau's TIGER road files.

  1. Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.

    PubMed

    Garcia, Matthew; Leonardi, Roberta; Zhang, Yong-Mei; Rehg, Jerold E; Jackowski, Suzanne

    2012-01-01

    Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B(5)) and controls the overall rate of coenzyme A (CoA) biosynthesis. Pank1 gene deletion in mice results in a metabolic phenotype where fatty acid oxidation and gluconeogenesis are impaired in the fasted state, leading to mild hypoglycemia. Inactivating mutations in the human PANK2 gene lead to childhood neurodegeneration, but Pank2 gene inactivation in mice does not elicit a phenotype indicative of the neuromuscular symptoms or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain.

  2. Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency

    PubMed Central

    Ghosh, Alok; Trivedi, Prachi P.; Timbalia, Shrishiv A.; Griffin, Aaron T.; Rahn, Jennifer J.; Chan, Sherine S. L.; Gohil, Vishal M.

    2014-01-01

    Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. PMID:24549041

  3. Cooperation between COA6 and SCO2 in COX2 maturation during cytochrome c oxidase assembly links two mitochondrial cardiomyopathies.

    PubMed

    Pacheu-Grau, David; Bareth, Bettina; Dudek, Jan; Juris, Lisa; Vögtle, F-Nora; Wissel, Mirjam; Leary, Scot C; Dennerlein, Sven; Rehling, Peter; Deckers, Markus

    2015-06-02

    Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.

  4. Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target. PMID:27676316

  5. SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase

    PubMed Central

    Laurent, Gaëlle; German, Natalie J.; Saha, Asish K.; de Boer, Vincent C. J.; Davies, Michael; Koves, Timothy R.; Dephoure, Noah; Fischer, Frank; Boanca, Gina; Vaitheesvaran, Bhavapriya; Lovitch, Scott B.; Sharpe, Arlene H.; Kurland, Irwin J.; Steegborn, Clemens; Gygi, Steven P.; Muoio, Deborah M.; Ruderman, Neil B.; Haigis, Marcia C.

    2013-01-01

    Summary Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a novel regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a novel SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis. PMID:23746352

  6. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  7. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  8. COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency

    PubMed Central

    Martinez Lyons, Anabel; Ardissone, Anna; Reyes, Aurelio; Robinson, Alan J; Moroni, Isabella; Fernandez-Vizarra, Erika; Zeviani, Massimo

    2016-01-01

    Background Assembly of cytochrome c oxidase (COX, complex IV, cIV), the terminal component of the mitochondrial respiratory chain, is assisted by several factors, most of which are conserved from yeast to humans. However, some of them, including COA7, are found in humans but not in yeast. COA7 is a 231aa-long mitochondrial protein present in animals, containing five Sel1-like tetratricopeptide repeat sequences, which are likely to interact with partner proteins. Methods Whole exome sequencing was carried out on a 19 year old woman, affected by early onset, progressive severe ataxia and peripheral neuropathy, mild cognitive impairment and a cavitating leukodystrophy of the brain with spinal cord hypotrophy. Biochemical analysis of the mitochondrial respiratory chain revealed the presence of isolated deficiency of cytochrome c oxidase (COX) activity in skin fibroblasts and skeletal muscle. Mitochondrial localization studies were carried out in isolated mitochondria and mitoplasts from immortalized control human fibroblasts. Results We found compound heterozygous mutations in COA7: a paternal c.410A>G, p.Y137C, and a maternal c.287+1G>T variants. Lentiviral-mediated expression of recombinant wild-type COA7 cDNA in the patient fibroblasts led to the recovery of the defect in COX activity and restoration of normal COX amount. In mitochondrial localization experiments, COA7 behaved as the soluble matrix protein Citrate Synthase. Conclusions We report here the first patient carrying pathogenic mutations of COA7, causative of isolated COX deficiency and progressive neurological impairment. We also show that COA7 is a soluble protein localized to the matrix, rather than in the intermembrane space as previously suggested. PMID:27683825

  9. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

    PubMed

    Peter, Dominik M; Vögeli, Bastian; Cortina, Niña Socorro; Erb, Tobias J

    2016-04-20

    Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories.

  10. Statin-like principles of bergamot fruit (Citrus bergamia): isolation of 3-hydroxymethylglutaryl flavonoid glycosides.

    PubMed

    Di Donna, Leonardo; De Luca, Giuseppina; Mazzotti, Fabio; Napoli, Anna; Salerno, Raffaele; Taverna, Domenico; Sindona, Giovanni

    2009-07-01

    The 3-hydroxy-3-methylglutaryl neohesperidosides of hesperetin (brutieridin, 1) and naringenin (melitidin, 2) were isolated and detected from the fruits of bergamot (Citrus bergamia). The structures of these compounds were determined by spectroscopic and chemical methods.

  11. The Role of Coa2 in Hemylation of Yeast Cox1 Revealed by Its Genetic Interaction with Cox10 ▿

    PubMed Central

    Bestwick, Megan; Khalimonchuk, Oleh; Pierrel, Fabien; Winge, Dennis R.

    2010-01-01

    Saccharomyces cerevisiae cells lacking the cytochrome c oxidase (CcO) assembly factor Coa2 are impaired in Cox1 maturation and exhibit a rapid degradation of newly synthesized Cox1. The respiratory deficiency of coa2Δ cells is suppressed either by the presence of a mutant allele of the Cox10 farnesyl transferase involved in heme a biosynthesis or through impaired proteolysis by the disruption of the mitochondrial Oma1 protease. Cox10 with an N196K substitution functions as a robust gain-of-function suppressor of the respiratory deficiency of coa2Δ cells but lacks suppressor activity for two other CcO assembly mutant strains, the coa1Δ and shy1Δ mutants. The suppressor activity of N196K mutant Cox10 is dependent on its catalytic function and the presence of Cox15, the second enzyme involved in heme a biosynthesis. Varying the substitution at Asn196 reveals a correlation between the suppressor activity and the stabilization of the high-mass homo-oligomeric Cox10 complex. We postulate that the mutant Cox10 complex has enhanced efficiency in the addition of heme a to Cox1. Coa2 appears to impart stability to the oligomeric wild-type Cox10 complex involved in Cox1 hemylation. PMID:19841065

  12. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Schnable, Patrick S.; Wen, Tsui-Jung

    2009-04-28

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  13. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2005-09-13

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  14. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2004-07-20

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.

  15. Insulin Signaling Regulates Fatty Acid Catabolism at the Level of CoA Activation

    PubMed Central

    Xu, Xiaojun; Gopalacharyulu, Peddinti; Seppänen-Laakso, Tuulikki; Ruskeepää, Anna-Liisa; Aye, Cho Cho; Carson, Brian P.; Mora, Silvia; Orešič, Matej; Teleman, Aurelio A.

    2012-01-01

    The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG) catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS). We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis. PMID:22275878

  16. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  17. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  18. A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

    PubMed

    Igarashi, Junsuke; Okamoto, Ryuji; Yamashita, Tetsuo; Hashimoto, Takeshi; Karita, Sakiko; Nakai, Kozo; Kubota, Yasuo; Takata, Maki; Yamaguchi, Fuminori; Tokuda, Masaaki; Sakakibara, Norikazu; Tsukamoto, Ikuko; Konishi, Ryoji; Hirano, Katsuya

    2016-03-01

    We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

  19. Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate

    PubMed Central

    Maloney, Finn P.; Gerwick, Lena; Gerwick, William H.; Sherman, David H.; Smith, Janet L.

    2016-01-01

    Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA. PMID:27573844

  20. Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

    PubMed Central

    Kassovska-Bratinova, S.; Fukao, T.; Song, X. Q.; Duncan, A. M.; Chen, H. S.; Robert, M. F.; Pérez-Cerdá, C.; Ugarte, M.; Chartrand, C.; Vobecky, S.; Kondo, N.; Mitchell, G. A.

    1996-01-01

    Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. Images Figure 2 Figure 6 PMID:8751852

  1. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  2. Discovery of Tumor-Specific Irreversible Inhibitors of Stearoyl CoA Desaturase

    PubMed Central

    Theodoropoulos, Panayotis C.; Gonzales, Stephen S.; Winterton, Sarah E.; Rodriguez-Navas, Carlos; McKnight, John S.; Morlock, Lorraine K.; Hanson, Jordan M.; Cross, Bethany; Owen, Amy E.; Duan, Yingli; Moreno, Jose R.; Lemoff, Andrew; Mirzaei, Hamid; Posner, Bruce A.; Williams, Noelle S.

    2016-01-01

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic to the same four of 12 human lung cancer cell lines at low nanomolar concentrations. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible stearoyl CoA desaturase (SCD) inhibitors. SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes were unable to activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors. PMID:26829472

  3. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  4. Spectroscopic Classification of ASASSN-16fn/AT2016coa and MASTER J202606.27-200732.6

    NASA Astrophysics Data System (ADS)

    Falco, E.; Calkins, M.; Challis, P.; Kirshner, R.; Prieto, J. L.; Stanek, K. Z.

    2016-06-01

    Optical spectra (range 350-760nm) of the supernova candidates ASASSN-16fn/AT2016coa (ATel #9081) and MASTER J202606.27-200732.6 (ATel #9056) were obtained on UT 2016 June 3 with the F. L. Whipple Observatory 1.5-m telescope (+ FAST).

  5. Carnitine palmitoyl transferase activity in Morris Hepatoma 7777 mitochondria and its sensitivity to malonyl CoA inhibition

    SciTech Connect

    Woldegiorgis, G.; Shrago, E.

    1986-05-01

    Earlier reports in the literature have indicated no detectable Carnitine Palymitoyl Transferase (CPT) activity in homogenates prepared from Morris Hepatoma 7777. In its study CPT activity in isolated mitochondria (mito) was measured by butanol extraction of the (/sup 3/H)palmitoyl carnitine formed as outlined by Bremer et al. Contrary to the earlier work where no appreciable activity of CPT was observed the authors find significant levels of CPT (2.6 nMol/min/mg protein) in isolated mito from Morris Hepatoma 7777 (MH 7777). The level of CPT activity observed in MH 7777 mito was, however, 36% lower compared to the host liver CPT activity (4.1 nMol/min/mg protein). The enzyme in MH 7777 mito showed 83% inhibition in the presence of 10 ..mu..M malonyl CoA, in agreement with the degree of sensitivity observed with the host liver isolated mito. On freeze thawing host mito, total CPT activity increased and the sensitivity of the enzyme to malonyl CoA decreased. Frozen thawed MH 7777 mito showed a similar response to malonyl CoA but no change in the total CPT level was observed. The authors results establish for the first time the presence of a malonyl CoA sensitive CPT in MH 7777 mito, which may have slightly different properties from normal due to the membrane environment of the enzyme.

  6. OUTCROP-BASED HIGH RESOLUTION GAMMA-RAY CHARACTERIZATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA). CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The COA supplies drinking water to a number of municipalities in central Oklahoma. Two major stratigraphic units in the COA, the Garber Sandstone and Wellington Formation, contain naturally occurring arsenic that exceeds government mandated drinking-water standards (EPA, 2001). ...

  7. Effects of HMG-COA Reductase Inhibitor Therapy on LDL Cholesterol Blood Levels in Hyperlipidemia: A Longitudinal Retrospective Anlaysis Using a Department of Defense Integrated Database.

    DTIC Science & Technology

    2007-11-02

    morbidity in hypercholesterolemic patients with angina pectoris or myocardial infarction.53඀ There were 4444 patients selected for inclusion and...A Randomized Trial in Patients with Previous Acute Myocardial Infarction and/or Unstable Angina Pectoris . American Journal of Cardiology 1995; 76...Patients with Previous Acute Myocardial Infarction and/or Unstable Angina Pectoris . American Journal of Cardiology 1995; 76:474-479. The

  8. Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W

    PubMed Central

    Grienenberger, Etienne; Kim, Sung Soo; Lallemand, Benjamin; Geoffroy, Pierrette; Heintz, Dimitri; Souza, Clarice de Azevedo; Heitz, Thierry; Douglas, Carl J.; Legrand, Michel

    2010-01-01

    The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors. PMID:21193572

  9. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  10. Model simulations of cooking organic aerosol (COA) over the UK using estimates of emissions based on measurements at two sites in London

    NASA Astrophysics Data System (ADS)

    Ots, Riinu; Vieno, Massimo; Allan, James D.; Reis, Stefan; Nemitz, Eiko; Young, Dominique E.; Coe, Hugh; Di Marco, Chiara; Detournay, Anais; Mackenzie, Ian A.; Green, David C.; Heal, Mathew R.

    2016-11-01

    Cooking organic aerosol (COA) is currently not included in European emission inventories. However, recent positive matrix factorization (PMF) analyses of aerosol mass spectrometer (AMS) measurements have suggested important contributions of COA in several European cities. In this study, emissions of COA were estimated for the UK, based on hourly AMS measurements of COA made at two sites in London (a kerbside site in central London and an urban background site in a residential area close to central London) for the full calendar year of 2012 during the Clean Air for London (ClearfLo) campaign. Iteration of COA emissions estimates and subsequent evaluation and sensitivity experiments were conducted with the EMEP4UK atmospheric chemistry transport modelling system with a horizontal resolution of 5 km × 5 km. The spatial distribution of these emissions was based on workday population density derived from the 2011 census data. The estimated UK annual COA emission was 7.4 Gg per year, which is an almost 10 % addition to the officially reported UK national total anthropogenic emissions of PM2.5 (82 Gg in 2012), corresponding to 320 mg person-1 day-1 on average. Weekday and weekend diurnal variation in COA emissions were also based on the AMS measurements. Modelled concentrations of COA were then independently evaluated against AMS-derived COA measurements from another city and time period (Manchester, January-February 2007), as well as with COA estimated by a chemical mass balance model of measurements for a 2-week period at the Harwell rural site (˜ 80 km west of central London). The modelled annual average contribution of COA to ambient particulate matter (PM) in central London was between 1 and 2 µg m-3 (˜ 20 % of total measured OA1) and between 0.5 and 0.7 µg m-3 in other major cities in England (Manchester, Birmingham, Leeds). It was also shown that cities smaller than London can have a central hotspot of population density of smaller

  11. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  12. Membrane remodeling, an early event in benzo[alpha]pyrene-induced apoptosis

    SciTech Connect

    Tekpli, Xavier; Rissel, Mary; Huc, Laurence; Catheline, Daniel; Sergent, Odile; Rioux, Vincent; Legrand, Philippe; Holme, Jorn A.; Dimanche-Boitrel, Marie-Therese; Lagadic-Gossmann, Dominique

    2010-02-15

    Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H{sub 2}O{sub 2} formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H{sub 2}O{sub 2} dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.

  13. Neuroprotective role for carbonyl reductase?

    PubMed

    Maser, Edmund

    2006-02-24

    Oxidative stress is increasingly implicated in neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, and Creutzfeld-Jakob diseases or amyotrophic lateral sclerosis. Reactive oxygen species seem to play a significant role in neuronal cell death in that they generate reactive aldehydes from membrane lipid peroxidation. Several neuronal diseases are associated with increased accumulation of abnormal protein adducts of reactive aldehydes, which mediate oxidative stress-linked pathological events, including cellular growth inhibition and apoptosis induction. Combining findings on neurodegeneration and oxidative stress in Drosophila with studies on the metabolic characteristics of the human enzyme carbonyl reductase (CR), it is clear now that CR has a potential physiological role for neuroprotection in humans. Several lines of evidence suggest that CR represents a significant pathway for the detoxification of reactive aldehydes derived from lipid peroxidation and that CR in humans is essential for neuronal cell survival and to confer protection against oxidative stress-induced brain degeneration.

  14. Acyl CoA Binding Proteins are Required for Cuticle Formation and Plant Responses to Microbes.

    PubMed

    Xia, Ye; Yu, Keshun; Gao, Qing-Ming; Wilson, Ella V; Navarre, Duroy; Kachroo, Pradeep; Kachroo, Aardra

    2012-01-01

    Fatty acids (FA) and lipids are well known regulators of plant defense. Our previous studies have shown that components of prokaryotic (plastidal) FA biosynthesis pathway regulate various aspects of plant defense. Here, we investigated the defense related roles of the soluble acyl CoA binding proteins (ACBPs), which are thought to facilitate the intracellular transport of FA/lipids. We show that ACBP3 and 4 are required for maintaining normal lipid levels and that ACBP3 contributes to the lipid flux between the prokaryotic and eukaryotic pathways. We also show that loss of ACBP3, 4, or 6 impair normal development of the cuticle and affect both basal and resistance protein-mediated defense against bacterial and fungal pathogens. Loss of ACBP3, 4, or 6 also inhibits the induction of systemic acquired resistance (SAR) due to the plants inability to generate SAR inducing signal(s). Together, these data show that ACBP3, ACBP4, and ACBP6 are required for cuticle development as well as defense against microbial pathogens.

  15. Real-Time Decision Support for Course of Action/Enemy Course of Action (COA/ECOA) Analysis

    DTIC Science & Technology

    2005-10-01

    used. 3. A domain ontology must be given. Based on these assumptions, we propose the CAFSIN solution, standing for COA Analysis based on Fuzzified...the Pentagon” and “DoD” are wired together as the same word and are represented as a single node). 2. Polysemy : words with different meanings in the...Given an ontology with these requirements satisfied, a standard hashing function may be used to directly identify a specific node in the ontology

  16. Correlation of ATP citrate lyase and acetyl CoA levels with trichothecene production in Fusarium graminearum.

    PubMed

    Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei

    2013-11-21

    The correlation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride.

  17. SUBSURFACE WELL-LOG CORRELATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA), CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The fluvial Garber Sandstone and the underlying Wellington Formation are important sources of drinking water in central Oklahoma. These formations, which make up much of the COA, consist of amalgamated sandstones with some interbedded mudstones, siltstones, and local mudstone- a...

  18. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... About half of these individuals adopt a male gender role in adolescence or early adulthood. Related Information ... 1730-5. Citation on PubMed Cohen-Kettenis PT. Gender change in 46,XY persons with 5alpha-reductase- ...

  19. Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes.

    PubMed

    Goepfert, Simon; Vidoudez, Charles; Tellgren-Roth, Christian; Delessert, Syndie; Hiltunen, J Kalervo; Poirier, Yves

    2008-12-01

    Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.

  20. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  1. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  2. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

  3. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  4. Coenzyme Q10 reverses mitochondrial dysfunction in atorvastatin-treated mice and increases exercise endurance.

    PubMed

    Muraki, Ayako; Miyashita, Kazutoshi; Mitsuishi, Masanori; Tamaki, Masanori; Tanaka, Kumiko; Itoh, Hiroshi

    2012-08-01

    Statins are cholesterol-lowering drugs widely used in the prevention of cardiovascular diseases; however, they are associated with various types of myopathies. Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and thus decrease biosynthesis of low-density lipoprotein cholesterol and may also reduce ubiquinones, essential coenzymes of a mitochondrial electron transport chain, which contain isoprenoid residues, synthesized through an HMG-CoA reductase-dependent pathway. Therefore, we hypothesized that statin treatment might influence physical performance through muscular mitochondrial dysfunction due to ubiquinone deficiency. The effect of two statins, atorvastatin and pravastatin, on ubiquinone content, mitochondrial function, and physical performance was examined by using statin-treated mice. Changes in energy metabolism in association with statin treatment were studied by using cultured myocytes. We found that atorvastatin-treated mice developed muscular mitochondrial dysfunction due to ubiquinone deficiency and a decrease in exercise endurance without affecting muscle mass and strength. Meanwhile, pravastatin at ten times higher dose of atorvastatin had no such effects. In cultured myocytes, atorvastatin-related decrease in mitochondrial activity led to a decrease in oxygen utilization and an increase in lactate production. Conversely, coenzyme Q(10) treatment in atorvastatin-treated mice reversed atorvastatin-related mitochondrial dysfunction and a decrease in oxygen utilization, and thus improved exercise endurance. Atorvastatin decreased exercise endurance in mice through mitochondrial dysfunction due to ubiquinone deficiency. Ubiquinone supplementation with coenzyme Q(10) could reverse atorvastatin-related mitochondrial dysfunction and decrease in exercise tolerance.

  5. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

    PubMed

    Henin, N; Vincent, M F; Gruber, H E; Van den Berghe, G

    1995-04-01

    AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

  6. The IFN-γ-induced Transcriptional Program of the CIITA Gene is Inhibited by Statins

    PubMed Central

    Lee, Sun Jung; Qin, Hongwei; Benveniste, Etty N.

    2009-01-01

    Summary Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-γ induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins, however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-γ-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-γ inducible expression of CIITA, and statins inhibit IFN-γ-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-γ induction of the CIITA gene is regulated by the transcription factors STAT-1α, IRF-1 and USF-1. We previously reported that statins inhibit constitutive STAT-1α expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1α and IRF-1 to the endogenous CIITA pIV promoter. The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA Polymerase II, p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene. PMID:18601229

  7. Increase in hepatic expression of SREBP-2 by gemfibrozil administration to rats.

    PubMed

    Roglans, N; Peris, C; Verd, J C; Alegret, M; Vázquez, M; Sánchez, R M; Laguna, J C

    2001-09-15

    It is well known that gemfibrozil increases the biliary output of cholesterol and phospholipids, but we have little knowledge about the impact these changes have on liver cholesterol and phospholipid biosynthetic pathways. In the present study, no changes were detected in liver lipids and CTP:phosphocholine cytidylyltransferase after gemfibrozil administration to rats. On the contrary, 3-hydroxy-3-methylglutaryl-CoA reductase mRNA (9.9-fold) and Rd activity (16.7-fold) and phosphatidate phosphohydrolase activity (1.7-fold) increased, while plasma apo B-cholesterol (40%) and triglyceride (43%) levels decreased. As a part of a compensatory homeostatic response, we report for the first time that gemfibrozil administration to rats increased the hepatic sterol regulatory element binding protein-2 (SREBP-2) mRNA (2.9-fold) and mature protein (2.2-fold) levels. An early increase in the transcriptional activity of SREBP-2 elicited by gemfibrozil administration might be responsible for the observed changes in HMG-CoA reductase, phosphatidate phosphohydrolase, and SREBP-2 expression.

  8. Lovastatin and perillyl alcohol inhibit glioma cell invasion, migration, and proliferation--impact of Ras-/Rho-prenylation.

    PubMed

    Afshordel, Sarah; Kern, Beatrice; Clasohm, Jasmin; König, Hildegard; Priester, Maike; Weissenberger, Jakob; Kögel, Donat; Eckert, Gunter P

    2015-01-01

    Alterations in small GTPase mediated signal transduction pathways have emerged as a central step in the molecular pathogenesis of glioblastoma (GBM), the most common malignant brain tumor in adults. Farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) are derived from mevalonate, whose production is catalyzed by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Prenylation by FPP and GGPP is required for membrane insertion and oncogenic function of Ras- and Rho-proteins, within the stimulation of the Ras-Raf-MEK-ERK pathway. A straightforward prediction from HMG-CoA reductase inhibitor studies is that statins decrease FPP and GGPP levels and diminish ERK signaling ensuring less proliferation and migration of cancer cells. Perillyl alcohol (POH), a naturally occurring monoterpene inhibits prenyltransferases and is able to inhibit cancer cell growth, but the underlying mechanism is still unclear. We here report that lovastatin (LOV) and POH impair the regulation of the mevalonate- and the Ras-Raf-MEK-ERK pathway in U87 and U343 glioblastoma cells. Both compounds affected the post-translational modification of H-Ras and Rac1. While LOV diminished the substrates of the transferase reaction that catalyze prenylation, POH inhibited the enzymes itself. Our data highlight the impact of isoprenoids for post-translational modification of small GTPases promoting proliferation, migration and invasion capabilities in glioma cells.

  9. Hydroxylation of compactin (ML-236B) by CYP105D7 (SAV_7469) from Streptomyces avermitilis.

    PubMed

    Yao, Qiuping; Ma, Li; Liu, Ling; Ikeda, Haruo; Fushinobu, Shinya; Li, Shengying; Xu, Lian-Hua

    2017-03-09

    Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the former shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products including pravastatin (hydroxylated at the C6 position). The steady state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The Km, and kcat values for compactin were 39.1 ± 8.8 μM and 1.12 ± 0.09 min⁻¹, respectively. The kcat/Km value for compactin (0.029 min⁻¹.μM⁻¹) was lower than that for diclofenac (0.114 min⁻¹.μM⁻¹). Spectroscopic analysis showed that CYP105D7 binds to compactin with a Kd value of 17.5 ± 3.6 μM. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.

  10. Unpredictable chronic mild stress induces anxiety and depression-like behaviors and inactivates AMP-activated protein kinase in mice.

    PubMed

    Zhu, Shenghua; Wang, Junhui; Zhang, Yanbo; Li, Victor; Kong, Jiming; He, Jue; Li, Xin-Min

    2014-08-12

    The unpredictable chronic mild stress (UCMS) model was developed based upon the stress-diathesis hypothesis of depression. Most effects of UCMS can be reversed by antidepressants, demonstrating a strong predictive validity of this model for depression. However, the mechanisms underlying the effects induced by UCMS remain incompletely understood. Increasing evidence has shown that AMP-activated protein kinase (AMPK) regulates intracellular energy metabolism and is especially important for neurons because neurons are known to have small energy reserves. Abnormalities in the AMPK pathway disturb normal brain functions and synaptic integrity. In the present study, we first investigated the effects of UCMS on a battery of different tests measuring anxiety and depression-like behaviors in female C57BL/6N mice after 4 weeks of UCMS exposure. Stressed mice showed suppressed body weight gain, heightened anxiety, and increased immobility in the forced swim and tail suspension tests. These results are representative of some of the core symptoms of depression. Simultaneously, we observed decrease of synaptic proteins in the cortex of mice subjected to UCMS, which is associated with decreased levels of phosphorylated AMP-activated protein kinase α (AMPKα) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). Our findings suggest that AMPKα inactivation might be a mechanism by which UCMS causes anxiety/depression-like behaviors in mice.

  11. Lovastatin for hypercholesterolemia.

    PubMed

    Zeller, F P; Uvodich, K C

    1988-01-01

    Lovastatin is the first 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor approved for the treatment of primary hypercholesterolemia. It is indicated as adjunctive therapy to dietary control and should be initiated at 20 mg/d in the evening. With higher dosages, twice-daily dosing is preferred, particularly when the dosage reaches the maximum recommended 80 mg/d. Compared with other drugs available, lovastatin has been shown to have good efficacy and a low incidence of side effects. Limited pharmacokinetic information available from the manufacturer reports absorption approximately 30 percent, protein binding greater than 95 percent, and a dual pathway for elimination through both urine (10 percent) and feces (83 percent). The drug has been clinically tested versus placebo and in combination with other cholesterol-lowering drugs. Lovastatin is effective in lowering total cholesterol and low-density lipoprotein cholesterol by 25-30 percent, with nonfamilial (hypercholesterolemic) patients responding better than those with the familial form of the disease. One percent of lovastatin patients have discontinued therapy because of intolerable side effects. The most common complaints are flatulence and diarrhea; more severe abnormalities include elevation of liver enzymes and an unclear propensity for producing lens opacities. The monthly cost to a patient taking 20 mg/d is approximately $44. Although the drug should be added to hospital formularies, long-term safety experience and competition from other HMG-CoA reductase inhibitors will determine lovastatin's final therapeutic role.

  12. Pectin isolated from prickly pear (Opuntia SSP) modifies LDL metabolism in cholesterol-fed guinea pigs

    SciTech Connect

    Fernandez, M.L.; McNamara, D.J. )

    1990-02-26

    The effects of dietary pectin on plasma and hepatic cholesterol (CH) levels, plasma lipoprotein profiles, hepatic 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase activity, and low density lipoprotein (LDL) binding to hepatic membranes were investigated by feeding 1% pectin to guinea pigs on a high CH diet. Animals were fed either chow + 0.25% CH (HC diet) or the CH diet + 1% prickly pear pectin (HC-P diet) for 25 days. Plasma CH levels were decreased 26% by the HC-P with 33% decreases in LDL and KDL. LDL peak density shifted from 1.040 to 1.055 g/ml with pectin. Hepatic total, free and esterified CH levels were reduced 60, 40 and 85% respectively by the HC-P diet. In contrast, HMG-CoA reductase activity was unaffected. {sup 125}I-LDL binding to hepatic membranes was increased by intake of the HC-P diet compared to the HC diet. The affinity of the apo B/E receptor for LDL was not affected by dietary pectin while the receptor number was increased 1.5-fold in animals on the HC-P diet. These data suggest that the parameters of HC metabolism affected by dietary pectin are consistent with an increased demand on the hepatic CH pools which possibly results from increased fecal excretion of bile acids.

  13. Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis.

    PubMed

    Schulz, Martin Michael Peter; Reisen, Felix; Zgraggen, Silvana; Fischer, Stephanie; Yuen, Don; Kang, Gyeong Jin; Chen, Lu; Schneider, Gisbert; Detmar, Michael

    2012-10-02

    Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.

  14. Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase.

    PubMed

    Boll, Matthias; Einsle, Oliver; Ermler, Ulrich; Kroneck, Peter M H; Ullmann, G Matthias

    2016-01-01

    In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry.

  15. Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

    PubMed Central

    Vick, Jacob E.; Clomburg, James M.; Blankschien, Matthew D.; Chou, Alexander; Kim, Seohyoung

    2014-01-01

    We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli. PMID:25527535

  16. Acetoacetyl Coenzyme A Reductase and Polyhydroxybutyrate Synthesis in Rhizobium (Cicer) sp. Strain CC 1192

    PubMed Central

    Chohan, Shahid N.; Copeland, Les

    1998-01-01

    Biochemical controls that regulate the biosynthesis of poly-3-hydroxybutyrate (PHB) were investigated in Rhizobium (Cicer) sp. strain CC 1192. This species is of interest for studying PHB synthesis because the polymer accumulates to a large extent in free-living cells but not in bacteroids during nitrogen-fixing symbiosis with chickpea (Cicer arietinum L.) plants. Evidence is presented that indicates that CC 1192 cells retain the enzymic capacity to synthesize PHB when they differentiate from the free-living state to the bacteroid state. This evidence includes the incorporation by CC 1192 bacteroids of radiolabel from [14C]malate into 3-hydroxybutyrate which was derived by chemically degrading insoluble material from bacteroid pellets. Furthermore, the presence of an NADPH-dependent acetoacetyl coenzyme A (CoA) reductase, which was specific for R-(−)-3-hydroxybutyryl-CoA and NADP+ in the oxidative direction, was demonstrated in extracts from free-living and bacteroid cells of CC 1192. Activity of this enzyme in the reductive direction appeared to be regulated at the biochemical level mainly by the availability of substrates. The CC 1192 cells also contained an NADH-specific acetoacetyl-CoA reductase which oxidized S-(+)-3-hydroxybutyryl-CoA. A membrane preparation from CC 1192 bacteroids readily oxidized NADH but not NADPH, which is suggested to be a major source of reductant for nitrogenase. Thus, a high ratio of NADPH to NADP+, which could enhance delivery of reductant to nitrogenase, could also favor the reduction of acetoacetyl-CoA for PHB synthesis. This would mean that fine controls that regulate the partitioning of acetyl-CoA between citrate synthase and 3-ketothiolase are important in determining whether PHB accumulates. PMID:9687441

  17. The aldo-keto reductase superfamily homepage.

    PubMed

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  18. Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases.

    PubMed

    Murphy, D G; Davidson, W S

    1986-01-01

    An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.

  19. Cholesterol metabolism and colon cancer.

    PubMed

    Broitman, S A; Cerda, S; Wilkinson, J

    1993-01-01

    While epidemiologic and concordant experimental data indicate a direct relationship between dietary fat (and presumably caloric) intake and the development of colon cancer, the effect of dietary cholesterol on this disease is still not clear. However, there appears to be a developing literature concerning an inverse relationship between serum and plasma cholesterol levels, and the risk for colon cancer. Findings that low serum cholesterol levels are apparent as early as ten years prior to the detection of colon cancer implies that sub clinical disease is probably not involved initially in this process. The possibility of low serum cholesterol as a bio-marker was considered in epidemiologic studies which focused upon obese men with lower than normal serum cholesterol levels who were found to be at increased risk to colon cancer. While the relationship between low serum cholesterol and colonic or intestinal cholesterol metabolism is presently not understood, current genetic studies provide a promising though as yet unexplored potential association. Alterations which occur during the developmental progression of colonic cancer include changes in chromosome 5, which also carries two genes vital to the biosynthesis and regulation of systemic and cellular cholesterol metabolism, 3-hydroxy-3-methylglutaryl coenzyme A synthase, and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA R). Regulation of cholesterol metabolism in intestinal cells in vivo and in vitro varies from that seen in normal fibroblasts or hepatocytes in terms of exogenous sources of cholesterol and how these sources regulate internal synthesis. Colonic cancer cells have been used to assess small bowel enterocyte cholesterol metabolism, which has been possible because of their ability to differentiate in culture, however information regarding true colonic enterocyte cholesterol metabolism is relatively scarce. Colonic cancer cells have been shown to possess a diminished or nonexistent ability to use

  20. Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase | Office of Cancer Genomics

    Cancer.gov

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease.

  1. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  2. The tyrosyl free radical in ribonucleotide reductase.

    PubMed Central

    Gräslund, A; Sahlin, M; Sjöberg, B M

    1985-01-01

    The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID:3007085

  3. Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells.

    PubMed

    Hasumi, K; Otsuki, R; Endo, A

    1985-08-01

    Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.

  4. Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

    PubMed

    Venkatraman, Janani; Bhat, Jyothi; Solapure, Suresh M; Sandesh, Jatheendranath; Sarkar, Debasmita; Aishwarya, Sundaram; Mukherjee, Kakoli; Datta, Santanu; Malolanarasimhan, Krishnan; Bandodkar, Balachandra; Das, Kaveri S

    2012-03-01

    The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.

  5. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  6. Isolation, sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase.

    PubMed

    Liu, G Y

    2009-09-01

    The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.

  7. C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight.

    PubMed

    Mera, Paula; Bentebibel, Assia; López-Viñas, Eduardo; Cordente, Antonio G; Gurunathan, Chandrashekaran; Sebastián, David; Vázquez, Irene; Herrero, Laura; Ariza, Xavier; Gómez-Puertas, Paulino; Asins, Guillermina; Serra, Dolors; García, Jordi; Hegardt, Fausto G

    2009-03-15

    Central nervous system administration of C75 produces hypophagia and weight loss in rodents identifying C75 as a potential drug against obesity and type 2 diabetes. However, the mechanism underlying this effect is unknown. Here we show that C75-CoA is generated chemically, in vitro and in vivo from C75 and that it is a potent inhibitor of carnitine palmitoyltranferase 1 (CPT1), the rate-limiting step of fatty-acid oxidation. Three-D docking and kinetic analysis support the inhibitory effect of C75-CoA on CPT1. Central nervous system administration of C75 in rats led to C75-CoA production, inhibition of CPT1 and lower body weight and food intake. Our results suggest that inhibition of CPT1, and thus increased availability of fatty acids in the hypothalamus, contribute to the pharmacological mechanism of C75 to decrease food intake.

  8. CoaTx-II, a new dimeric Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom with bactericidal potential: Insights into its structure and biological roles.

    PubMed

    Almeida, J R; Lancellotti, M; Soares, A M; Calderon, L A; Ramírez, D; González, W; Marangoni, S; Da Silva, S L

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria.

  9. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  10. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  11. Rutin inhibits oleic acid induced lipid accumulation via reducing lipogenesis and oxidative stress in hepatocarcinoma cells.

    PubMed

    Wu, Cheng-Hsun; Lin, Ming-Cheng; Wang, Hsueh-Chun; Yang, Mon-Yuan; Jou, Ming-Jia; Wang, Chau-Jong

    2011-03-01

    Excessive lipid accumulation within liver has been proposed to cause obesity, hyperlipidemia, diabetes, and fatty liver disease. Rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, has various biological functions, including antioxidant, anti-inflammatory, and anticancer effects. However, a hypolipidemic effect of rutin on fatty liver disease has not been reported. In this study, we examined the effect of rutin on reducing lipid accumulation in hepatic cells. Hepatocytes were treated with oleic acid (OA) containing with or without rutin to observe the lipid accumulation by Nile red stain. The result showed rutin suppressed OA-induced lipid accumulation and increased adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activity in hepatocytes. The expression of critical molecule involved in lipid synthesis, sterol regulatory element binding proteins-1 (SREBP-1), was attenuated in rutin-treated cells. Moreover, long-term incubation of rutin inhibited the transcriptions of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), glycerol-3-phosphate acyltransferase (GPAT), fatty acid synthase (FAS), and acetyl-coenzyme carboxylase (ACC). Besides, we also found out the antioxidative effect of rutin by increasing the expression of peroxisome proliferator-activated receptor (PPAR)-α and antioxidative enzymes. Taken together, our findings suggest rutin could attenuate lipid accumulation by decreasing lipogenesis and oxidative stress in hepatocyte.

  12. Elicitation of Diosgenin Production in Trigonella foenum-graecum (Fenugreek) Seedlings by Methyl Jasmonate

    PubMed Central

    Chaudhary, Spandan; Chikara, Surendra K.; Sharma, Mahesh C.; Chaudhary, Abhinav; Alam Syed, Bakhtiyar; Chaudhary, Pooja S.; Mehta, Aditya; Patel, Maulik; Ghosh, Arpita; Iriti, Marcello

    2015-01-01

    The effects of methyl jasmonate (MeJA), an elicitor of plant defense mechanisms, on the biosynthesis of diosgenin, a steroidal saponin, were investigated in six fenugreek (Trigonella foenum-graecum) varieties (Gujarat Methi-2, Kasuri-1, Kasuri-2, Pusa Early Branching, Rajasthan Methi and Maharashtra Methi-5). Treatment with 0.01% MeJA increased diosgenin levels, in 12 days old seedlings, from 0.5%–0.9% to 1.1%–1.8%. In addition, MeJA upregulated the expression of two pivotal genes of the mevalonate pathway, the metabolic route leading to diosgenin: 3-hydroxy-3-methylglutaryl-CoA reductase (HMG) and sterol-3-β-glucosyl transferase (STRL). In particular, MeJA increased the expression of HMG and STRL genes by 3.2- and 22.2-fold, respectively, in the Gujarat Methi-2 variety, and by 25.4- and 28.4-fold, respectively, in the Kasuri-2 variety. Therefore, MeJA may be considered a promising elicitor for diosgenin production by fenugreek plants. PMID:26694357

  13. Effects of rosuvastatin and pitavastatin on ischemia-induced myocardial stunning in dogs.

    PubMed

    Satoh, Kumi; Takaguri, Akira; Itagaki, Mai; Kano, Seiichiro; Ichihara, Kazuo

    2008-04-01

    Incomplete recovery of myocardial contraction after reperfusion following brief ischemia is called the "stunning phenomenon" in an animal experiment. A hydrophilic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) does not affect this phenomenon, but lipophilic statins further reduce the contraction during reperfusion. The effects of novel hydrophilic rosuvastatin and lipophilic pitavastatin on myocardial stunning in dogs were examined. In a preliminary experiment in vitro, pitavastatin reduced L6 cell viability at 10(-6) M and higher, whereas rosuvastatin and pravastatin up to 10(-5) M did not show such effects. An empty capsule or a capsule filled with rosuvastatin (2 mg/kg per day) or pitavastatin (0.4 mg/kg per day) was orally administered to dogs. After 3 weeks, both statins lowered the serum cholesterol level to the same extent. Under pentobarbital anesthesia, dogs were subjected to 15-min ischemia followed by 120-min reperfusion. Ischemia arrested the myocardial contraction in the ischemic area, and reperfusion recovered it but incompletely, showing the stunning phenomenon. Rosuvastatin did not modify the stunning phenomenon, while pitavastatin further deteriorated the myocardial contraction during reperfusion.

  14. Simvastatin induces insulin resistance in L6 skeletal muscle myotubes by suppressing insulin signaling, GLUT4 expression and GSK-3β phosphorylation.

    PubMed

    Yaluri, Nagendra; Modi, Shalem; Kokkola, Tarja

    2016-11-11

    Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used for the treatment of hypercholesterolemia. Recent data indicates that simvastatin increases the risk of new-onset diabetes by impairing both insulin secretion and insulin sensitivity. However, systematic evaluation of mechanistic pathways is lacking. We aimed to explore the effects of simvastatin on glucose uptake and underlying mechanisms using L6 skeletal muscle myotubes. We performed our experiments at basal and insulin-stimulated conditions, at normal (5.5 mM) and high (16.7 mM) glucose. We showed that simvastatin inhibited glucose uptake at all conditions. We also found out that pravastatin, another widely used statin with different physicochemical properties, did not inhibit glucose uptake. The effect of simvastatin was reversed with geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate, implying that reduced protein geranylgeranylation has a role in simvastatin-induced insulin resistance. Simvastatin also decreased phosphorylation of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), AKT and glycogen synthase kinase 3β (GSK-3β), and downregulated GLUT4. In conclusion, our data indicate that simvastatin decreased both basal and insulin-stimulated glucose uptake through inhibiting the critical steps in IR/IRS-1/AKT signaling cascade, and by hindering GLUT4 function and normal regulation of glycogen synthesis, contributing to insulin resistance.

  15. Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle.

    PubMed

    van Vliet, A K; Nègre-Aminou, P; van Thiel, G C; Bolhuis, P A; Cohen, L H

    1996-11-08

    Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 +/- 6 nM and 4.0 +/- 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 +/- 38 nM). Through inhibition of mevalonate production, these compounds have a distinct inhibiting effect on cell proliferation. Because proliferation of myoblasts is important in the repair of damaged skeletal muscle, experiments were performed to investigate the effect of lovastatin, simvastatin, and pravastatin on cell proliferation and cell viability. The more potent inhibitors of sterol synthesis, lovastatin, and simvastatin, were able to inhibit the proliferation of these cells during 3 days of incubation with drug concentrations of 1 microM for lovastatin and 0.1 microM or 1 microM for simvastatin. DNA synthesis was decreased by more than 80% in the presence of 1 microM of lovastatin or simvastatin. In contrast, under these conditions, pravastatin had no influence on cell proliferation or DNA synthesis, which is probably related to the lack of inhibition of sterol synthesis by pravastatin on extended incubation. The three 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors did not disturb cell viability because mitochondrial dehydrogenase activity and ATP content remained proportional to the number of cells in the culture at any concentration used.

  16. The Vitamin E Analog Gamma-Tocotrienol (GT3) and Statins Synergistically Up-Regulate Endothelial Thrombomodulin (TM)

    PubMed Central

    Pathak, Rupak; Ghosh, Sanchita P.; Zhou, Daohong; Hauer-Jensen, Martin

    2016-01-01

    Statins; a class of routinely prescribed cholesterol-lowering drugs; inhibit 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR) and strongly induce endothelial thrombomodulin (TM); which is known to have anti-inflammatory; anti-coagulation; anti-oxidant; and radioprotective properties. However; high-dose toxicity limits the clinical use of statins. The vitamin E family member gamma-tocotrienol (GT3) also suppresses HMGCR activity and induces TM expression without causing significant adverse side effects; even at high concentrations. To investigate the synergistic effect of statins and GT3 on TM; a low dose of atorvastatin and GT3 was used to treat human primary endothelial cells. Protein-level TM expression was measured by flow cytometry. TM functional activity was determined by activated protein C (APC) generation assay. Expression of Kruppel-like factor 2 (KLF2), one of the key transcription factors of TM, was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). TM expression increased in a dose-dependent manner after both atorvastatin and GT3 treatment. A combined treatment of a low-dose of atorvastatin and GT3 synergistically up-regulated TM expression and functional activity. Finally; atorvastatin and GT3 synergistically increased KLF2 expression. These findings suggest that combined treatment of statins with GT3 may provide significant health benefits in treating a number of pathophysiological conditions; including inflammatory and cardiovascular diseases. PMID:27869747

  17. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

    PubMed

    Li, Huan-Jun; He, Yi-Long; Zhang, De-Huai; Yue, Tong-Hui; Jiang, Lu-Xi; Li, Na; Xu, Jun-Wei

    2016-06-10

    The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8μg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

  18. Association of HMGCR polymorphism with late-onset Alzheimer's disease in Han Chinese

    PubMed Central

    Tan, Meng-Shan; Wang, Hui-Fu; Tan, Chen-Chen; Zhang, Wei; Zheng, Zhan-Jie; Kong, Ling-Li; Wang, Zi-Xuan; Jiang, Teng; Yu, Jin-Tai; Tan, Lan

    2016-01-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) acts as a potential genetic modifier for Alzheimer's disease (AD). Previous reports identified that HMGCR rs3846662 polymorphism is associated with biosynthesis of cholesterol in AD pathology. In order to assess the involvement of the HMGCR polymorphism in the risk of late-onset AD (LOAD) in northern Han Chinese, we performed a case–control study of 2334 unrelated subjects (984 cases and 1350 age- and gender-matched controls) to evaluate the genotype and allele distributions of the HMGCR rs3846662 with LOAD. The genotype distribution (GG, AG, AA) of rs3846662 was significantly different between LOAD patients and controls (P = 0.003), but the allele distribution did not reach a significant difference (P = 0.614). After adjusting for age, gender and the APOE ε4 status, the minor A allele of rs3846662 was validated as a protective factor for LOAD in dominant model (OR = 0.796, P = 0.02, 95% CI = 0.657–0.965). Interestingly, we observed rs3846662 polymorphism was only significantly associated with LOAD in APOE ε4 non-carriers (OR = 0.735, P = 0.005, 95% CI = [0.593, 0.912]). In conclusion, our study demonstrates A allele of HMGCR rs3846662 acts as a protective factor for LOAD in northern Han Chinese. PMID:27009838

  19. Aspirin and clopidogrel response variability: review of the published literature.

    PubMed

    Ferguson, Angela D; Dokainish, Hisham; Lakkis, Nasser

    2008-01-01

    Antiplatelet resistance has been proposed as a possible mechanism to explain recurrent cardiovascular events in patients who have coronary artery disease and who are undergoing dual antiplatelet therapy. A comprehensive search on PubMed was conducted for literature that was printed in the English language between January 1996 and November 2007 on aspirin and clopidogrel resistance. Significant traits for aspirin hyporesponsiveness were female sex, older age, and lower levels of hemoglobin. Diabetes mellitus and elevated body mass index showed trends toward a higher incidence of resistance in some aspirin trials but did not reach statistical significance. Clopidogrel studies suggested that patients with type-2 diabetes mellitus are more likely to manifest inadequate response to the medication. Although 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors were initially suspected to decrease response to clopidogrel, later studies refuted this possibility. Patients with a suboptimal response to aspirin or clopidogrel seem to be at increased risk of recurrent cardiovascular events. Large clinical trials with standardized laboratory methods and well-defined protocols are needed to determine whether common features exist in patients with suspected hyporesponsiveness to antiplatelet therapy, and to validate the clinical relevance of response variability. A concise nonarbitrary definition of physiologic "resistance" is needed, and investigators should identify patients as having a variable response to antiplatelet therapy.

  20. Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

    PubMed

    Yu, Xuya; Ji, Sen-Lin; He, Yi-Long; Ren, Meng-Fei; Xu, Jun-Wei

    2014-01-01

    We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

  1. Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

    PubMed

    Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

    2011-10-01

    In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

  2. Role of statins in the treatment of multiple sclerosis.

    PubMed

    Ciurleo, Rosella; Bramanti, Placido; Marino, Silvia

    2014-09-01

    Statins as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase are widely prescribed for hypercholesterolemia treatment. In the last years, statins have also been shown to exert immunomodulatory and anti-inflammatory effects which appear to be related to inhibition of isoprenylation of small GTP-binding proteins and, at least in part, independent of their cholesterol-lowering effects. These "pleiotropic" effects make statins an attractive treatment option for immune-mediated disorders such as multiple sclerosis. Studies in vitro and in experimental autoimmune encephalomyelitis animal model seem to support not only the efficacy of statins as immunomodulatory agents but also their potential neuroprotective properties, although the exact mechanism with which statins exert these effects has not yet been fully understood. The immunomodulatory, anti-inflammatory and neuroprotective properties of statins provided the incentive for several clinical trials in multiple sclerosis, in which they were tested not only as mono-therapy but also in combination with interferon-β. However, the attempt to translate the results of animal model studies in humans produced conflicting results. Further large, prospective, randomized, double-blind, placebo-controlled trials, designed to evaluate the long-term effects of statins alone or in add-on to other disease-modifying therapies, are needed to support their routine clinical use in multiple sclerosis.

  3. Statins meditate anti-atherosclerotic action in smooth muscle cells by peroxisome proliferator-activated receptor-γ activation.

    PubMed

    Fukuda, Kazuki; Matsumura, Takeshi; Senokuchi, Takafumi; Ishii, Norio; Kinoshita, Hiroyuki; Yamada, Sarie; Murakami, Saiko; Nakao, Saya; Motoshima, Hiroyuki; Kondo, Tatsuya; Kukidome, Daisuke; Kawasaki, Shuji; Kawada, Teruo; Nishikawa, Takeshi; Araki, Eiichi

    2015-01-30

    The peroxisome proliferator-activated receptor-γ (PPARγ) is an important regulator of lipid and glucose metabolism, and its activation is reported to suppress the progression of atherosclerosis. We have reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) activate PPARγ in macrophages. However, it is not yet known whether statins activate PPARγ in other vascular cells. In the present study, we investigated whether statins activate PPARγ in smooth muscle cells (SMCs) and endothelial cells (ECs) and thus mediate anti-atherosclerotic effects. Human aortic SMCs (HASMCs) and human umbilical vein ECs (HUVECs) were used in this study. Fluvastatin and pitavastatin activated PPARγ in HASMCs, but not in HUVECs. Statins induced cyclooxygenase-2 (COX-2) expression in HASMCs, but not in HUVECs. Moreover, treatment with COX-2-siRNA abrogated statin-mediated PPARγ activation in HASMCs. Statins suppressed migration and proliferation of HASMCs, and inhibited lipopolysaccharide-induced expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in HASMCs. These effects of statins were abrogated by treatment with PPARγ-siRNA. Treatment with statins suppressed atherosclerotic lesion formation in Apoe(-/-) mice. In addition, transcriptional activity of PPARγ and CD36 expression were increased, and the expression of MCP-1 and TNF-α was decreased, in the aorta of statin-treated Apoe(-/-) mice. In conclusion, statins mediate anti-atherogenic effects through PPARγ activation in SMCs. These effects of statins on SMCs may be beneficial for the prevention of atherosclerosis.

  4. Effects of dietary tannic acid on the growth, hepatic gene expression, and antioxidant enzyme activity in Brandt's voles (Microtus brandti).

    PubMed

    Ye, Man-Hong; Nan, Yan-Lei; Ding, Meng-Meng; Hu, Jun-Bang; Liu, Qian; Wei, Wan-Hong; Yang, Sheng-Mei

    2016-01-01

    This study was designed to investigate the physiological and biochemical responses of Brandt's voles to the persistent presence of dietary tannic acid. The diet for animals in the experimental group was supplemented with 3% dietary tannic acid for 5weeks. The control group received a commercial lab chow. No significant differences were detected in body weight, organ (heart, kidney, and liver) weights, and organ parameters between animals from two groups. However, voles in the experimental group had significantly higher daily food intake, increased contents of proline and histidine in saliva and feces after protein hydrolysis, and elevated hepatic expression of transferrin than the control. Our results suggested the existence of adaptive strategies developed in Brandt's voles to overcome the adverse effects of dietary tannic acid. (1) Food consumption was increased to satisfy their nutritional demands. (2) The secretion of tannic-acid-binding salivary proteins was promoted. (3) The absorption of iron was enhanced. These alterations contributed to neutralize the negative effects of tannic acid and maintain body mass in animals supplemented with tannic acid. As the result of the consumption of tannic acid, hepatic expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase was significantly decreased, while the overall potential of the antioxidant system, characterized by increased hepatic enzymatic activities of catalase and glutathione peroxidase, was enhanced. Our results also implied the involvement of tannic acid in the regulation of lipid metabolism and oxidative stress in voles.

  5. Effect of mevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes.

    PubMed

    Liu, Xiaomu; You, Wei; Cheng, Haijian; Zhang, Qingfeng; Song, Enliang; Wan, Fachun; Han, Hong; Liu, Guifen

    2016-02-01

    Mevalonic acid (MVA) is a key material in the synthesis of cholesterol; indeed, intracellular cholesterol synthesis is also called the mevalonic acid pathway. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in cholesterol biosynthesis. This study suggests that MVA may play an important role in the differentiation of bovine adipose tissue in vivo. We investigated differential mRNA expression in bovine intramuscular preadipocytes (BIPs) and bovine subcutaneous preadipocytes (BSPs) by culturing cells from the longissimus dorsi muscle and subcutaneous fat tissues of Luxi yellow cattle. The morphology of lipid accumulation of bovine preadipocytes was detected by Oil Red O staining, and total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol (HDLC) levels were measured. Temporospatial expression of HMGR was investigated by real-time quantitative polymerase chain reaction (PCR). The TC, LDLC, and HDLC content did not significantly differ over time but increased slowly with increasing MVA concentration. HMGR expression increased over time and with increasing concentrations of MVA. MVA increased adipose cell proliferation in a dose-dependent and time-dependent manner. MVA stimulated HMGR expression in two cell types and its influence on adipocyte differentiation.

  6. Downregulation of miR-150 Expression by DNA Hypermethylation Is Associated with High 2-Hydroxy-(4-methylthio)butanoic Acid-Induced Hepatic Cholesterol Accumulation in Nursery Piglets.

    PubMed

    Jia, Yimin; Ling, Mingfa; Zhang, Luchu; Jiang, Shuxia; Sha, Yusheng; Zhao, Ruqian

    2016-10-12

    Excess 2-hydroxy-(4-methylthio)butanoic acid (HMB) supplementation induces hyperhomocysteinemia, which contributes to hepatic cholesterol accumulation. However, it is unclear whether and how high levels of HMB break hepatic cholesterol homeostasis in nursery piglets. In this study, HMB oversupplementation suppressed food intake and decreased body weight in nursery piglets. Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in HMB groups. Accordingly, HMB significantly increased the protein content of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine N-methyltransferase (GNMT) but decreased that of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1). Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p targeting the 3'-untranslated regions (UTRs) of GNMT and HMGCR was detected in the liver of HMB-treated piglets, and their functional validation was confirmed by dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter was detected in association with suppressed miR-150 expression in the livers of HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol unhomeostasis by dietary methyl donor supplementation.

  7. Effect of coenzyme q10 on myopathic symptoms in patients treated with statins.

    PubMed

    Caso, Giuseppe; Kelly, Patricia; McNurlan, Margaret A; Lawson, William E

    2007-05-15

    Treatment of hypercholesterolemia with statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) is effective in the primary and secondary prevention of cardiovascular disease. However, statin use is often associated with a variety of muscle-related symptoms or myopathies. Myopathy may be related in part to statin inhibition of the endogenous synthesis of coenzyme Q10, an essential cofactor for mitochondrial energy production. The aim of this study is to determine whether coenzyme Q10 supplementation would reduce the degree of muscle pain associated with statin treatment. Patients with myopathic symptoms were randomly assigned in a double-blinded protocol to treatment with coenzyme Q10 (100 mg/day, n = 18) or vitamin E (400 IU/day, n = 14) for 30 days. Muscle pain and pain interference with daily activities were assessed before and after treatment. After a 30-day intervention, pain severity decreased by 40% (p <0.001) and pain interference with daily activities decreased by 38% (p <0.02) in the group treated with coenzyme Q10. In contrast, no changes in pain severity (+9%, p = NS) or pain interference with daily activities (-11%, p = NS) was observed in the group treated with vitamin E. In conclusion, results suggest that coenzyme Q10 supplementation may decrease muscle pain associated with statin treatment. Thus, coenzyme Q10 supplementation may offer an alternative to stopping treatment with these vital drugs.

  8. Efficacy of statins on sirtuin 1 and endothelial nitric oxide synthase expression: the role of sirtuin 1 gene variants in human coronary atherosclerosis.

    PubMed

    Kilic, Ulkan; Gok, Ozlem; Elibol-Can, Birsen; Uysal, Omer; Bacaksiz, Ahmet

    2015-04-01

    Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and are used to reduce the risk of coronary artery disease (CAD) due to their pleiotropic effects. Recently, greater focus has been placed on the role of sirtuin 1 (SIRT1) in cardiovascular disease research. However, insufficient data exist on the relationships between statins, SIRT1 protein levels, and SIRT1 gene variants. In the present study, we investigated the effects of statins, atorvastatin and rosuvastatin, in CAD patients by analysing the associations between SIRT1 gene variants, rs7069102C>G and rs2273773C>T, and SIRT1/endothelial nitric oxide (eNOS) expression, as well as total antioxidant and oxidant status, and the oxidative stress index. SIRT1 expression was significantly higher, and eNOS expression was significantly lower in CAD patients when compared with controls. Statin treatment reduced SIRT1 expression and increased eNOS expression, similar to the levels found in the control population, independent from the studied SIRT1 gene variants. Oxidative stress parameters were significantly increased in CAD patients, and were decreased by statin treatment, demonstrating the antioxidative effects of statins on atherosclerosis. These results indicate that statin treatment could produce its protective effect on cardiovascular disease through the inhibition of SIRT1 expression. This is the first study reporting on the effect of statins, specifically atorvastatin and rosuvastatin, on SIRT1 expression in CAD patients.

  9. Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes.

    PubMed

    Li, Weihua; Liang, Xiaojing; Zeng, Zhipeng; Yu, Kaizhen; Zhan, Shaopeng; Su, Qiang; Yan, Yinzhi; Mansai, Huseen; Qiao, Weitong; Yang, Qi; Qi, Zhongquan; Huang, Zhengrong

    2016-10-01

    Simvastatin,a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, is clinically used in the prevention and treatment of cardiovascular diseases. Numerous studies demonstrate that statins increase the risk of new-onset diabetes in long-term therapy, but mechanisms underpinning this effect are still unclear. Here, we investigated whether simvastatin inhibited the glucose uptake activity and the underlying mechanisms in C2C12 myotubes. Our studies showed that simvastatin significantly inhibited glucose uptake activity and GLUT4 translocation, whereas the effect was reversible with mevalonolactone (ML), which acts as an intermediate of cholesterol synthesis pathway. Mechanistically, the inhibition of glucose uptake and GLUT4 translocation elicited by simvastatin were associated with the suppression of the insulin receptor (IR)/IR substrate (IRS)/Akt signaling cascade. Simvastatin suppressed the phosphorylation of IR, IRS-1 and Akt, and total expression of IR or IRS-1, but did not affect Akt. Furthermore, simvastatin decreased Rac1 GTP binding. In conclusion, our findings indicate that simvastatin suppresses glucose uptake activity and GLUT4 translocation via IR-dependent IRS-1/PI3K/Akt pathway. These results provide an important new insight into the mechanism of statins on insulin sensitivity which may be associated with new-onset diabetes.

  10. Lipidomic-based investigation into the regulatory effect of Schisandrin B on palmitic acid level in non-alcoholic steatotic livers.

    PubMed

    Kwan, Hiu Yee; Niu, Xuyan; Dai, Wenlin; Tong, Tiejun; Chao, Xiaojuan; Su, Tao; Chan, Chi Leung; Lee, Kim Chung; Fu, Xiuqiong; Yi, Hua; Yu, Hua; Li, Ting; Tse, Anfernee Kai Wing; Fong, Wang Fun; Pan, Si-Yuan; Lu, Aiping; Yu, Zhi-Ling

    2015-03-13

    Schisandrin B (SchB) is one of the most abundant bioactive dibenzocyclooctadiene derivatives found in the fruit of Schisandra chinensis. Here, we investigated the potential therapeutic effects of SchB on non-alcoholic fatty-liver disease (NAFLD). In lipidomic study, ingenuity pathway analysis highlighted palmitate biosynthesis metabolic pathway in the liver samples of SchB-treated high-fat-diet-fed mice. Further experiments showed that the SchB treatment reduced expression and activity of fatty acid synthase, expressions of hepatic mature sterol regulatory element binding protein-1 and tumor necrosis factor-α, and hepatic level of palmitic acid which is known to promote progression of steatosis to steatohepatitis. Furthermore, the treatment also reduced hepatic fibrosis, activated nuclear factor-erythroid-2-related factor-2 which is known to attenuate the progression of NASH-related fibrosis. Interestingly, in fasting mice, a single high-dose SchB induced transient lipolysis and increased the expressions of adipose triglyceride lipase and phospho-hormone sensitive lipase. The treatment also increased plasma cholesterol levels and 3-hydroxy-3-methylglutaryl-CoA reductase activity, reduced the hepatic low-density-lipoprotein receptor expression in these mice. Our data not only suggest SchB is a potential therapeutic agent for NAFLD, but also provided important information for a safe consumption of SchB because SchB overdosed under fasting condition will have adverse effects on lipid metabolism.

  11. Synergistic effects of ultraviolet-B and methyl jasmonate on tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

    PubMed

    Wang, Cong Hui; Zheng, Li Ping; Tian, Hao; Wang, Jian Wen

    2016-06-01

    Tanshinones are major bioactive diterpenoids of Salvia miltiorrhiza roots used for the treatment of cardiocerebral diseases. To develop effective elicitation and bioprocess strategies for the enhanced production of tanshinones, ultraviolet-B (UV-B) irradiation and methyl jasmonate (MeJA) elicitation were applied alone or in combination respectively in S. miltiorrhiza hairy root cultures. Our results showed 40-min UV-B irradiation at 40μW/cm(2) stimulated tanshinone production without any suppression of root growth, suggesting a new effective elicitor to S. miltiorrhiza hairy root cultures for tanshinone production. Moreover, the combined treatment of UV-B irradiation and MeJA exhibited synergistic effects on the expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (SmHMGR) and geranylgeranyl diphosphate synthase (SmGGPPS) genes in the tanshinone biosynthetic pathway. When hairy roots of 18-day-old cultures were exposed to the combined elicitation for 9days, the maximum production of tanshinone reached to 28.21mg/L, a 4.9-fold increase over the control. The combined elicitation of UV-B and MeJA was firstly used to stimulate the production of biologically important secondary metabolites in hairy root cultures.

  12. PCSK9 inhibitors: A new era of lipid lowering therapy

    PubMed Central

    Chaudhary, Rahul; Garg, Jalaj; Shah, Neeraj; Sumner, Andrew

    2017-01-01

    Hyperlipidemia is a well-established risk factor for developing cardiovascular disease (CVD). The recent American College of Cardiology and American Heart Association guidelines on lipid management emphasize treatment of individuals at increased risk for developing CVD events with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) at doses proven to reduce CVD events. However, there are limited options for patients who are either intolerant to statin therapy, develop CVD despite being on maximally tolerated statin therapy, or have severe hypercholesterolemia. Recently the Food and Drug Administration approved two novel medications for low-density lipoprotein (LDL)-cholesterol reduction: Evolocumab and Alirocumab. These agents target and inactivate proprotein convertase subtilsin-kexin type 9 (PCSK9), a hepatic protease that attaches and internalizes LDL receptors into lysosomes hence promoting their destruction. By preventing LDL receptor destruction, LDL-C levels can be lowered 50%-60% above that achieved by statin therapy alone. This review explores PCSK-9 biology and the mechanisms available to alter it; clinical trials targeting PCSK9 activity, and the current state of clinically available inhibitors of PCSK9. PMID:28289523

  13. Targeted Drug Delivery to Treat Pain and Cerebral Hypoxia

    PubMed Central

    Davis, Thomas P.

    2013-01-01

    Limited drug penetration is an obstacle that is often encountered in treatment of central nervous system (CNS) diseases including pain and cerebral hypoxia. Over the past several years, biochemical characteristics of the brain (i.e., tight junction protein complexes at brain barrier sites, expression of influx and efflux transporters) have been shown to be directly involved in determining CNS permeation of therapeutic agents; however, the vast majority of these studies have focused on understanding those mechanisms that prevent drugs from entering the CNS. Recently, this paradigm has shifted toward identifying and characterizing brain targets that facilitate CNS drug delivery. Such targets include the organic anion–transporting polypeptides (OATPs in humans; Oatps in rodents), a family of sodium-independent transporters that are endogenously expressed in the brain and are involved in drug uptake. OATP/Oatp substrates include drugs that are efficacious in treatment of pain and/or cerebral hypoxia (i.e., opioid analgesic peptides, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors). This clearly suggests that OATP/Oatp isoforms are viable transporter targets that can be exploited for optimization of drug delivery to the brain and, therefore, improved treatment of CNS diseases. This review summarizes recent knowledge in this area and emphasizes the potential that therapeutic targeting of OATP/Oatp isoforms may have in facilitating CNS drug delivery and distribution. Additionally, information presented in this review will point to novel strategies that can be used for treatment of pain and cerebral hypoxia. PMID:23343976

  14. Statin Use in Prostate Cancer: An Update

    PubMed Central

    Babcook, Melissa A.; Joshi, Aditya; Montellano, Jeniece A.; Shankar, Eswar; Gupta, Sanjay

    2016-01-01

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, known as statins, are commonly prescribed for the treatment of hypercholesterolemia and cardiovascular disease. A systematic review was conducted using the keywords “statin and prostate cancer” within the title search engines including PubMed, Web of Science, and the Cochrane Library for relevant research work published between 2004 and December 2015. Although still premature, accumulating clinical evidence suggests that statin use may be beneficial in the prevention and/or treatment of prostate cancer. These human studies consist of meta-analyses of secondary endpoints obtained from randomized, controlled cardiovascular disease clinical trials of statins, patient database, observational studies, and a few, small case–control studies, directly addressing statin use on prostate cancer pathology and recurrence. This review summarizes and discusses the recent clinical literature on statins and prostate cancer with a recommendation to move forward with randomized, placebo-controlled clinical trials, investigating the use of statins. Additional preclinical testing of statins on prostate cancer cell lines and in vivo models is needed to elucidate pathways and determine its efficacy for prevention and/or treatment of prostate cancer, more specifically, the difference in the effectiveness of lipophilic versus hydrophilic statins in prostate cancer. PMID:27441003

  15. Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress

    PubMed Central

    Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M. M.; Pandey, Rakesh

    2017-01-01

    Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress. PMID:28157221

  16. Protection against osteoporosis by statins is linked to a reduction of oxidative stress and restoration of nitric oxide formation in aged and ovariectomized rats.

    PubMed

    Yin, Hong; Shi, Zhen-Guo; Yu, Yong-Sheng; Hu, Jing; Wang, Ru; Luan, Zhi-Peng; Guo, Dai-Hong

    2012-01-15

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, have been used as a cholesterol-lowering drug to treat hyperlipidemia clinically. In recent years, accumulating evidence indicates the possible beneficial effect of statins on osteoporosis. The aim of present study was to investigate whether protection against osteoporosis by statins is linked to a reduction of oxidative stress and restoration of nitric oxide (NO) formation in aged and ovariectomized rats. The aged and ovariectomized rats were used as two models of osteoporosis for evaluation of the effect of simvastatin. It was found that simvastatin abated oxidative stress, increased NO production, subsequently attenuating osteoporosis in two models. In the in vitro studies, the protective effects against H(2)O(2)-induced cell injury were examined in the MG-63 human osteoblastic cells. It was found that simvastatin ameliorated H(2)O(2)-induced cell loss and cell apoptosis and increased alkaline phosphatase (ALP) activity in osteoblastic cells. Simvastatin abated oxidative stress through enhancing catalase, heme oxygenase 1 (HO-1), and superoxide dismutase (SOD) activity and suppressing NADPH oxidase activity. In addition, simvastatin raised nitric oxide synthase (NOS) activity and eNOS expression at basal condition; inhibited NOS activity and iNOS expression when treated with H(2)O(2). In conclusion, protection against osteoporosis by statins is linked to a reduction of oxidative stress and restoration of NO formation in aged and ovariectomized rats.

  17. Statins inhibit pulmonary artery smooth muscle cell proliferation by upregulation of HO-1 and p21WAF1.

    PubMed

    Li, Manxiang; Liu, Yuan; Shi, Hongyang; Zhang, Yonghong; Wang, Guizuo; Xu, Jing; Lu, Jiamei; Zhang, Dexin; Xie, Xinming; Han, Dong; Wu, Yuanyuan; Li, Shaojun

    2012-10-01

    Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which has been shown to ameliorate the development of pulmonary hypertension in animal model by suppression of pulmonary artery smooth muscle cells (PASMCs) proliferation, yet its underlying molecular mechanisms are not completely understood. In this study, we show that simvastatin dose-dependently inhibited serotonin-stimulated PASMCs proliferation. This was accompanied with the parallel induction of heme oxyganase-1 (HO-1) and upregulation of p21(WAF1). More importantly, we found that Tin-protoporphyrin (SnPP), a selective inhibitor of HO-1, could block the effect of simvastatin on inhibition of cell proliferation in response to serotonin and abolish simvastatin-induced p21(WAF1) expression. The inhibitive effect of simvastatin on cell proliferation was also significantly suppressed by silencing p21(WAF1) with siRNA transfection. The extent of effect of SnPP on inhibition of cell proliferation was similar to that of lack of p21(WAF1) by siRNA transfection. Taken together, our study suggests that simvastatin inhibits PASMCs proliferation by sequential upregulation of HO-1 and p21(WAF1) to benefit pulmonary hypertension.

  18. A novel peroxisome proliferator-activated receptor alpha/gamma dual agonist demonstrates favorable effects on lipid homeostasis.

    PubMed

    Guo, Qiu; Sahoo, Soumya P; Wang, Pei-Ran; Milot, Denise P; Ippolito, Marc C; Wu, Margaret S; Baffic, Joanne; Biswas, Chhabi; Hernandez, Melba; Lam, My-Hanh; Sharma, Neelam; Han, Wei; Kelly, Linda J; MacNaul, Karen L; Zhou, Gaochao; Desai, Ranjit; Heck, James V; Doebber, Thomas W; Berger, Joel P; Moller, David E; Sparrow, Carl P; Chao, Yu-Sheng; Wright, Samuel D

    2004-04-01

    Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-alpha/gamma activities. In keeping with its PPARgamma activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPARalpha or PPARalpha/gamma agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPARgamma agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPARalpha/gamma agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.

  19. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism.

    PubMed

    Wang, Wenxian; Wong, Chi-Wai

    2010-03-01

    Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) serves as an inducible coactivator for a number of transcription factors to control energy metabolism. Insulin signaling through Akt kinase has been demonstrated to phosphorylate PGC-1alpha at serine 571 and downregulate its activity in the liver. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors that reduce cholesterol synthesis in the liver. In this study, we found that statins reduced the active form of Akt and enhanced PGC-1alpha activity. Specifically, statins failed to activate an S571A mutant of PGC-1alpha. The activation of PGC-1alpha by statins selectively enhanced the expression of energy metabolizing enzymes and regulators including peroxisome proliferator-activated receptor alpha, acyl-CoA oxidase, carnitine palmitoyl transferase-1A, and pyruvate dehydrogenase kinase 4. Importantly, a constitutively active form of Akt partially reduced the statin-enhanced gene expression. Our study thus provides a plausible mechanistic explanation for the hypolipidemic effect of statin through elevating the rate of beta-oxidation and mitochondrial Kreb's cycle capacity to enhance fatty acid utilization while reducing the rate of glycolysis.

  20. Atorvastatin increases hepatic fatty acid beta-oxidation in sucrose-fed rats: comparison with an MTP inhibitor.

    PubMed

    Funatsu, Toshiyuki; Kakuta, Hirotoshi; Takasu, Toshiyuki; Miyata, Keiji

    2002-11-29

    We investigated the effects of atorvastatin, a widely used 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and BMS-201038, a microsomal triglyceride transfer protein (MTP) inhibitor, in sucrose-fed hypertriglyceridemic rats to determine whether the activation of beta-oxidation by these compounds plays a role in their hypotriglyceridemic effect. The decrease in plasma triglyceride concentration and post-Triton very low-density lipoprotein (VLDL) triglyceride concentration, a measure of hepatic triglyceride secretion, by atorvastatin (30 mg/kg p.o.) for 2 weeks was to approximately the same degree as those by BMS-201038 (0.3 mg/kg). Atorvastatin (30 mg/kg) increased hepatic beta-oxidation activity by 54% (P < 0.01), while BMS-201038 (0.3 mg/kg) had no significant effect. Atorvastatin decreased hepatic triglyceride, fatty acid and cholesteryl ester concentrations by 21% to 39%, whereas BMS-201038 increased these variables by 28% to 307%. In the atorvastatin-treated groups, a significant relationship was seen not only between hepatic beta-oxidation activity and hepatic triglyceride concentration (R(2) = 0.535, P < 0.01), but also between hepatic and plasma triglyceride concentrations (R(2) = 0.586, P < 0.01). No effect of atorvastatin on hepatic fatty acid synthesis was observed. These results indicate that the activation of hepatic beta-oxidation by atorvastatin may contribute to the decrease in hepatic triglyceride concentration, leading to its hypotriglyceridemic effect.

  1. Is Statin Use Associated With Tendon Rupture? A Population-Based Retrospective Cohort Analysis.

    PubMed

    Contractor, Tahmeed; Beri, Abhimanyu; Gardiner, Joseph C; Tang, Xiaoqin; Dwamena, Francesca C

    2015-01-01

    Previous case reports and small studies have suggested that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (HMG-CoA-Is) may increase the risk of tendon rupture. We conducted a population-based retrospective cohort evaluation to better assess this relationship. From approximately 800,000 enrollees of a private insurance database, those who were aged ≤64 years with at least 1 year of continuous enrollment were selected. Exposure was defined as initiation of HMG-CoA-I after the beginning of the study period. Each exposed person was matched with 2 controls of similar age and gender. Baseline characteristics, including known risk factors for tendon rupture, were compared between exposed and control cohorts with fidelity to the study's matched design. After adjusting for differences in follow-up and baseline characteristics, incidence rate ratios for tendon rupture was assessed in HMG-CoA-I users and nonusers. A total of 34,749 exposed patients were matched with 69,498 controls. There was no difference in the occurrence of tendon ruptures in HMG-CoA-I users versus nonusers. The results remained unchanged after adjustment for age and gender. In conclusion, this population-based retrospective cohort evaluation suggests that use of HMG-CoA-Is as a group are not associated with tendon rupture.

  2. Simvastatin Treatment Highlights a New Role for the Isoprenoid/Cholesterol Biosynthetic Pathway in the Modulation of Emotional Reactivity and Cognitive Performance in Rats

    PubMed Central

    Segatto, Marco; Manduca, Antonia; Lecis, Claudio; Rosso, Pamela; Jozwiak, Adam; Swiezewska, Ewa; Moreno, Sandra; Trezza, Viviana; Pallottini, Valentina

    2014-01-01

    The aim of the present work was to shed light on the role played by the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and memory consolidation in rodents through the inhibition of the key and rate-limiting enzyme 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR) both in vivo and in vitro with simvastatin. Three-month-old male Wistar rats treated for 21 days with simvastatin or vehicle were tested in the social interaction, elevated plus-maze, and inhibitory avoidance tasks; after behavioral testing, the amygdala, hippocampus, prefrontal cortex, dorsal, and ventral striatum were dissected out for biochemical assays. In order to delve deeper into the molecular mechanisms underlying the observed effects, primary rat hippocampal neurons were used. Our results show that HMGR inhibition by simvastatin induces anxiogenic-like effects in the social interaction but not in the elevated plus-maze test, and improves memory consolidation in the inhibitory avoidance task. These effects are accompanied by imbalances in the activity of specific prenylated proteins, Rab3 and RhoA, involved in neurotransmitter release, and synaptic plasticity, respectively. Taken together, the present findings indicate that the isoprenoid/cholesterol biosynthetic pathway is critically involved in the physiological modulation of both emotional and cognitive processes in rodents. PMID:24108067

  3. Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro.

    PubMed

    Cartocci, Veronica; Segatto, Marco; Di Tunno, Ilenia; Leone, Stefano; Pfrieger, Frank W; Pallottini, Valentina

    2016-09-01

    During differentiation, neurons acquire their typical shape and functional properties. At present, it is unclear, whether this important developmental step involves metabolic changes. Here, we studied the contribution of the mevalonate (MVA) pathway to neuronal differentiation using the mouse neuroblastoma cell line N1E-115 as experimental model. Our results show that during differentiation, the activity of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR), a key enzyme of MVA pathway, and the level of Low Density Lipoprotein receptor (LDLr) decrease, whereas the level of LDLr-related protein-1 (LRP1) and the dimerization of Scavanger Receptor B1 (SRB-1) rise. Pharmacologic inhibition of HMGR by simvastatin accelerated neuronal differentiation by modulating geranylated proteins. Collectively, our data suggest that during neuronal differentiation, the activity of the MVA pathway decreases and we postulate that any interference with this process impacts neuronal morphology and function. Therefore, the MVA pathway appears as an attractive pharmacological target to modulate neurological and metabolic symptoms of developmental neuropathologies. J. Cell. Biochem. 117: 2036-2044, 2016. © 2016 Wiley Periodicals, Inc.

  4. SUGP1 is a novel regulator of cholesterol metabolism

    PubMed Central

    Kim, Mee J.; Yu, Chi-Yi; Theusch, Elizabeth; Naidoo, Devesh; Stevens, Kristen; Kuang, Yu-Lin; Schuetz, Erin; Chaudhry, Amarjit S.; Medina, Marisa W.

    2016-01-01

    A large haplotype on chromosome 19p13.11 tagged by rs10401969 in intron 8 of SURP and G patch domain containing 1 (SUGP1) is associated with coronary artery disease (CAD), plasma LDL cholesterol levels, and other energy metabolism phenotypes. Recent studies have suggested that TM6SF2 is the causal gene within the locus, but we postulated that this locus could harbor additional CAD risk genes, including the putative splicing factor SUGP1. Indeed, we found that rs10401969 regulates SUGP1 exon 8 skipping, causing non-sense-mediated mRNA decay. Hepatic Sugp1 overexpression in CD1 male mice increased plasma cholesterol levels 20–50%. In human hepatoma cell lines, SUGP1 knockdown stimulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) alternative splicing and decreased HMGCR transcript stability, thus reducing cholesterol synthesis and increasing LDL uptake. Our results strongly support a role for SUGP1 as a novel regulator of cholesterol metabolism and suggest that it contributes to the relationship between rs10401969 and plasma cholesterol. PMID:27206982

  5. Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

    PubMed

    Coria, Mariela J; Carmona Viglianco, Yamila V; Marra, Carlos A; Gomez-Mejiba, Sandra E; Ramirez, Dario C; Anzulovich, Ana C; Gimenez, Maria S

    2012-01-01

    Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

  6. Current and emerging treatments for hypercholesterolemia: A focus on statins and proprotein convertase subtilisin/kexin Type 9 inhibitors for perioperative clinicians

    PubMed Central

    Trentman, Terrence L.; Avey, Steven G.; Ramakrishna, Harish

    2016-01-01

    Statins are a mainstay of hyperlipidemia treatment. These drugs inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase and have beneficial effects on atherosclerosis including plaque stabilization, reduction of platelet activation, and reduction of plaque proliferation and inflammation. Statins also have a benefit beyond atherosclerotic plaque, including anticoagulation, vasodilatation, antioxidant effects, and reduction of mediators of inflammation. In the perioperative period, statins appear to contribute to improved outcomes via these mechanisms. Both vascular and nonvascular surgery patients have been shown in prospective studies to have lower risk of adverse cardiac outcomes when initiated on statins preoperatively. However, not all patients can tolerate statins; the search for novel lipid-lowering therapies led to the discovery of the proprotein convertase subtilisin/kexin Type 9 (PCSK9) inhibitors. These drugs are fully-humanized, injectable monoclonal antibodies. With lower PCSK9 activity, low-density lipoprotein cholesterol (LDL-C) receptors are more likely to be recycled to the hepatocyte surface, where they serve to clear plasma LDL-C. Evidence from several prospective studies shows that these new agents can significantly lower LDL-C levels. While PCSK9 inhibitors offer hope of effective therapy for patients with familial hyperlipidemia or intolerance of statins, several important questions remain, including the results of long term cardiovascular outcome studies. The perioperative effects of new LDL-C-lowering drugs are unknown at present but are likely to be similar to the older agents. PMID:28096572

  7. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway.

    PubMed

    Bhattacharya, Bonhi S; Sweby, Peter K; Minihane, Anne-Marie; Jackson, Kim G; Tindall, Marcus J

    2014-05-21

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

  8. Pectin isolated from prickly pear (Opuntia sp.) modifies low density lipoprotein metabolism in cholesterol-fed guinea pigs.

    PubMed

    Fernandez, M L; Trejo, A; McNamara, D J

    1990-11-01

    The effect of prickly pear soluble fiber on low density lipoprotein (LDL) metabolism was investigated by feeding male guinea pigs either a nonpurified diet containing 0.25% cholesterol (HC diet) or the HC diet + 1% prickly pear pectin (HC-P diet). Plasma cholesterol levels were significantly decreased by the HC-P diet, with a 33% decrease in LDL levels (p less than 0.02) and an increase in LDL density. Hepatic free and esterified cholesterol levels were reduced 40 and 85%, respectively (p less than 0.002), by the HC-P diet. Hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase levels were not different. 125I-LDL binding to hepatic membranes was increased 1.7-fold by the HC-P diet (p less than 0.001), with receptor affinity (Kd) being unaltered and receptor number (Bmax) being significantly increased (p less than 0.001). These data suggest that prickly pear pectin may act by a mechanism similar to that of bile acid-binding resins in lowering plasma cholesterol levels. The observed reduction in LDL and hepatic cholesterol levels and increase in LDL density and hepatic apolipoprotein B/E receptors are responses suggesting an increased demand on hepatic cholesterol from increased excretion of bile acids and interruption of the enterohepatic circulation.

  9. Prickly pear (Opuntia sp.) pectin reverses low density lipoprotein receptor suppression induced by a hypercholesterolemic diet in guinea pigs.

    PubMed

    Fernandez, M L; Lin, E C; Trejo, A; McNamara, D J

    1992-12-01

    The effects of prickly pear pectin on plasma LDL metabolism were investigated by feeding guinea pigs either a diet containing 15 g/100 g lard and 0.25 g/100 g cholesterol (LC diet) or the LC diet in which cellulose was partially replaced (2.5 g/100 g) by prickly pear pectin (LC-P diet). The LC-P diet lowered plasma LDL cholesterol concentrations by 33% (P < 0.001). Low density lipoprotein composition was modified by intake of prickly pear pectin; the relative percentages of free and esterified cholesterol were lower and triglycerides were higher in LDL from animals fed the LC-P diet (P < 0.05). Intake of prickly pear pectin did not affect hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity; however, hepatic free and esterified cholesterol concentrations were lowered by 46 and 64%, respectively. Hepatic apolipoprotein B/E receptor expression (Bmax) was 60% higher in animals fed the LC-P diet (P < 0.01). Similar to the in vitro data, receptor-mediated LDL fractional catabolic rates were 190% higher in animals fed the LC-P diet (P < 0.05), whereas apolipoprotein LDL flux rates were not affected. Apolipoprotein LDL pool size and fractional catabolic rates exhibited a significant correlation (r = -0.52, P < 0.01). These data indicate that an increase in apolipoprotein B/E receptor expression is a major metabolic response by which intake of prickly pear pectin decreases plasma LDL concentrations.

  10. Chronic psychosocial stress in male mice causes an up-regulation of scavenger receptor class B type 1 protein in the adrenal glands.

    PubMed

    Füchsl, Andrea M; Uschold-Schmidt, Nicole; Reber, Stefan O

    2013-07-01

    Mice exposed to chronic subordinate colony housing (CSC, 19 days) show an exaggerated adrenal corticosterone response to an acute heterotypic stressor (elevated platform (EPF), 5 min) despite no difference from EPF-exposed single-housed control (SHC) mice in corticotropin (ACTH) secretion. In the present study, we asked the question whether this CSC-induced increase in adrenal capability to produce and secrete corticosterone is paralleled by an enhanced adrenal availability and/or mobilization capacity of the corticosterone precursor molecule cholesterol. Employing oil-red staining and western blot analysis we revealed comparable relative density of cortical lipid droplets and relative protein expression of hormone-sensitive lipase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density lipoprotein receptor (LDL-R) between CSC and SHC mice. However, relative protein expression of the scavenger receptor class B type 1 (SR-BI) was increased following CSC exposure. Moreover, analysis of plasma high-density lipoprotein-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) revealed increased LDL-C levels in CSC mice. Together with the pronounced increase in adrenal weight, evidently mediated by hyperplasia of adrenocortical cells, these data strongly indicate an enhanced adrenal availability of and capacity to mobilize cholesterol in chronic psychosocially-stressed mice, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

  11. Hypolipidemic activity of okra is mediated through inhibition of lipogenesis and upregulation of cholesterol degradation.

    PubMed

    Wang, Hong; Chen, Gu; Ren, Dandan; Yang, Shang-Tian

    2014-02-01

    Little is known about the hypolipidemic activity of okra; therefore, we investigated the hypolipidemic activity of okra and its interaction with gene expression of several key components involved in lipid homeostasis. Male C57BL/6 mice were randomly divided into three groups and fed with hyperlipidemic diet or two hyperlipidemic diets supplemented with 1% or 2% okra powder for eight weeks. Results demonstrated that okra dose-dependently decreased serum and hepatic total cholesterol and triglyceride, and enhanced fecal excretion of bile acids. Gene expression analysis revealed that okra upregulated cholesterol 7α-hydroxylase (CYP7A1) expression, downregulated expression of sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthase (FAS), with no effect on sterol regulatory element-binding protein 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), low-density lipoprotein receptor (LDLR) and carnitine palmitoyltransferase-1A (CPT1A). It was suggested that hypolipidemic activity of okra was mediated most likely by upregulation of cholesterol degradation through CYP7A1 and by inhibition of lipogenesis through SREBP1c and FAS. Okra raw and fractionated polysaccharide showed strong bile acid binding capacity in vitro, which may contribute to the hypolipidemic activity observed. In conclusion, okra has potential application in the management of hyperlipidemia and its associated metabolic disorders.

  12. Statin therapy in the treatment of Alzheimer disease: what is the rationale?

    PubMed

    DeKosky, Steven T

    2005-12-01

    Alzheimer disease (AD) is a chronic neurodegenerative disorder that is manifested by cognitive decline, neuropsychiatric symptoms, and diffuse structural abnormalities in the brain. Its prevalence is predicted to rise 4-fold in the next 50 years. AD is characterized pathologically by deposition of extracellular beta-amyloid and accumulation of neurofibrillary tangles. Neuronal death and specific neurotransmitter deficits also are part of the pathologic picture. Strategies to delay symptom progression have focused on addressing the neurotransmitter deficits. Strategies to delay the onset or biologic progression of AD largely have targeted the plaques formed by the deposition of beta-amyloid. AD and cardiovascular disease share common risk factors, notably hypercholesterolemia, and occur together more often than expected by chance. Therapy with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) is the first-line treatment option for hypercholesterolemia, and observational studies have suggested that the risk of AD is reduced in patients who receive statin therapy in midlife. This reduction in risk of AD observed with statin therapy may be due to statins reducing beta-amyloid formation and deposition or to their known anti-inflammatory effects. Two randomized double-blind statin trials in patients with AD to assess the potential for statins to slow disease progression are currently under way. If successful, statin AD primary prevention trials may be developed.

  13. Utilization of digital differential display to identify differentially expressed genes related to rumen development.

    PubMed

    Kato, Daichi; Suzuki, Yutaka; Haga, Satoshi; So, KyoungHa; Yamauchi, Eri; Nakano, Miwa; Ishizaki, Hiroshi; Choi, Kichoon; Katoh, Kazuo; Roh, Sang-Gun

    2016-04-01

    This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)-based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5-week-old pre-weaning: n = 3; 15-week-old weaned calves: n = 6). Among the 11 genes, only 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), aldo-keto reductase family 1, member C1-like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre- and post-weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both.

  14. Dietary cholesterol supplementation improves growth and behavioral response of pigs selected for genetically high and low serum cholesterol.

    PubMed

    Schoknecht, P A; Ebner, S; Pond, W G; Zhang, S; McWhinney, V; Wong, W W; Klein, P D; Dudley, M; Goddard-Finegold, J; Mersmann, H J

    1994-02-01

    We hypothesized that, in pigs selected for low (L) or high (H) serum cholesterol for four generations, neonatal endogenous cholesterol synthesis would be sufficient to meet requirements for brain and body growth. In Experiment 1, eight 16-wk-old L pigs received a diet with or without 200 mg cholesterol/100 g diet for 35 d. Supplemented pigs grew approximately 25% faster and had a significantly greater concentration of free cholesterol in the cerebrum. In Experiment 2, 16 H and 16 L newborn pigs were fed a milk replacer with or without 200 mg cholesterol/100 g diet for 28 d. Pigs fed cholesterol had greater average daily gain (P < or = 0.09), significantly reduced liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, and significantly increased cerebral cholesterol content than pigs not fed cholesterol. One of three indices of exploratory behavior was significantly greater in the L pigs that received cholesterol compared with L pigs that did not receive cholesterol. These data suggest that these neonatal pigs are unable to produce sufficient cholesterol to meet requirements for normal growth and brain development and are dependent on dietary cholesterol in milk.

  15. [Microbial metabolites that inhibit sterol biosynthesis, their chemical diversity and characteristics of mode of action].

    PubMed

    Trenin, A S

    2013-01-01

    Inhibitors of sterol biosynthesis (ISB) are widespread in nature and characterized by appreciable diversity both in their chemical structure and mode of action. Many of these inhibitors express noticeable biological activity and approved themselves in development of various pharmaceuticals. In this review there is a detailed description of biologically active microbial metabolites with revealed chemical structure that have ability to inhibit sterol biosynthesis. Inhibitors of mevalonate pathway in fungous and mammalian cells, exhibiting hypolipidemic or antifungal activity, as well as inhibitors of alternative non-mevalonate (pyruvate gliceraldehyde phosphate) isoprenoid pathway, which are promising in the development of affective antimicrobial or antiparasitic drugs, are under consideration in this review. Chemical formulas of the main natural inhibitors and their semi-synthetic derivatives are represented. Mechanism of their action at cellular and biochemical level is discussed. Special attention is given to inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase (group of lovastatin) and inhibitors of acyl-CoA-cholesterol-acyl transferase (ACAT) that possess hypolipidemic activity and could be affective in the treatment of atherosclerosis. In case of inhibitors of late stages of sterol biosynthesis (after squalene formation) special attention is paid to compounds possessing evident antifungal and antitumoral activity. Explanation of mechanism of anticancer and antiviral action of microbial ISB, as well as the description of their ability to induce apoptosis is given.

  16. Statins Inhibit the Proliferation and Induce Cell Death of Human Papilloma Virus Positive and Negative Cervical Cancer Cells

    PubMed Central

    Crescencio, María Elena; Rodríguez, Emma; Páez, Araceli; Masso, Felipe A.; Montaño, Luis F.; López-Marure, Rebeca

    2009-01-01

    Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, have anti-tumoral effects on multiple cancer types; however, little is known about their effect on cervical cancer. We evaluated the effect on proliferation, cell cycle, oxidative stress and cell death of three statins on CaSki, HeLa (HPV+) and ViBo (HPV−) cervical cancer cell lines. Cell proliferation was assayed by crystal violet staining, cell cycle by flow cytometry and cell death by annexin-V staining. Reactive oxygen species (ROS) production was evaluated by the oxidation of 2,7-dichlorofluorescein diacetate and nitrite concentration (an indirect measure of nitric oxide (NO) production), by the Griess reaction. Inhibition of cell proliferation by atorvastatin, fluvastatin and simvastatin was dose-dependent. ViBo cells were the most responsive. Statins did not affect the cell cycle, instead they induced cell death. The antiproliferative effect in ViBo cells was completely inhibited with mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) treatments. In contrast, cell proliferation of CaSki and HeLa cells was partially (33%) rescued with these intermediates. The three statins increased ROS and nitrite production, mainly in the ViBo cell line. These results suggest that statins exert anti-tumoral effects on cervical cancer through inhibition of cell proliferation and induction of cell death and oxidative stress. Statins could be an aid in the treatment of cervical cancer, especially in HPV− tumors. PMID:23675166

  17. Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes.

    PubMed

    Michael, D R; Moss, J W E; Calvente, D Lama; Garaiova, I; Plummer, S F; Ramji, D P

    2016-06-01

    Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies.

  18. Plant Sterols: Diversity, Biosynthesis, and Physiological Functions.

    PubMed

    Valitova, J N; Sulkarnayeva, A G; Minibayeva, F V

    2016-08-01

    Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3-methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.

  19. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking.

    PubMed

    Islam, Barira; Sharma, Charu; Adem, Abdu; Aburawi, Elhadi; Ojha, Shreesh

    2015-01-01

    Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP(+)). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (-)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure-function relationship studies.

  20. Unmodified low density lipoprotein causes cholesteryl ester accumulation in J774 macrophages.

    PubMed

    Tabas, I; Weiland, D A; Tall, A R

    1985-01-01

    Cholesteryl ester (CE)-loaded macrophages (foam cells) are a prominent feature of atherosclerotic plaques. Previous studies have shown that human monocytes or resident mouse peritoneal macrophages accumulate CE in response to low density lipoprotein (LDL) only when the LDL has been appropriately chemically modified. By contrast, we report here that J774 macrophages accumulate large amounts of CE when incubated with unmodified LDL. The CE is stored in oil red O-positive droplets, which have the typical appearance of foam cell inclusions by electron microscopy. The fatty acid moieties of the cellular CE are enriched in oleate unlike those of LDL-CE, which are enriched in linoleate, indicating that the LDL-CE undergoes hydrolysis and reesterification by acyl CoA:cholesterol acyltransferase. Studies with 125I-labeled LDL at both 4 degrees C and 37 degrees C indicate that the LDL is internalized by a specific receptor that has several characteristics in common with the apolipoprotein B/E (apo B/E) receptor. However, in comparison with fibroblasts, the LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in J774 cells are relatively resistant to down-regulation by LDL or 25-hydroxycholesterol, leading to receptor-mediated CE storage. In addition, J774 cells appear to accumulate CE from LDL internalized by nonspecific means. Thus, macrophage-like cells can accumulate CE in response to unmodified LDL by both nonspecific and receptor-mediated processes.

  1. Statin safety: an appraisal from the adverse event reporting system.

    PubMed

    Davidson, Michael H; Clark, John A; Glass, Lucas M; Kanumalla, Anju

    2006-04-17

    The adverse event (AE) profiles of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) agents are of great interest, in particular the most recently approved statin, rosuvastatin. The forwarding of reports of AEs has been shown to be influenced by several reporting biases, including secular trend, the new drug reporting effect, product withdrawals, and publicity. Comparative assessments that use AE reporting rates are difficult to interpret under these circumstances, because such effects can themselves lead to marked increases in AE reporting. Consequently, many comparative reporting rate analyses are best carried out in conjunction with other metrics that put reporting burden into context, such as report proportion. All-AE reporting rates showed a temporal profile that resembled those of other statins when marketing cycle and secular trend were taken into account. A before-and-after cerivastatin withdrawal comparison showed a substantial increase in the reporting of AEs of interest for the statin class overall. Report proportion analyses indicated that the burden of rosuvastatin-associated AEs was similar to that for other statin agents. Analyses of monthly reporting rates showed that the reporting of rosuvastatin-associated rhabdomyolysis and renal failure have increased following AE-specific mass media publicity. Postrosuvastatin AE reporting patterns were comparable to those seen with other statins and did not resemble cerivastatin.

  2. Preclinical Activity of Simvastatin Induces Cell Cycle Arrest in G1 via Blockade of Cyclin D-Cdk4 Expression in Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Liang, Yu-Wei; Chang, Chi-Chang; Hung, Chao-Ming; Chen, Tzu-Yu; Huang, Tzuu-Yuan; Hsu, Yi-Chiang

    2013-01-01

    Lung cancer is the most common cause of cancer-related death. Nonetheless, a decrease in overall incidence and mortality has been observed in the last 30 years due to prevention strategies and improvements in the use of chemotherapeutic agents. In recent studies, Simvastatin (SIM) has demonstrated anti-tumor activity, as well as potent chemopreventive action. As an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), SIM has been shown to stimulate apoptotic cell death. In this study, an MTT assay revealed the cytotoxic activity of SIM against human large cell lung cancer (Non-small cell lung cancer; NSCLC) cells (NCI-H460); however, induced apoptosis was not observed in NCI-H460 cells. Protein expression levels of cell cycle regulating proteins Cdk4, Cyclin D1, p16 and p27 were markedly altered by SIM. Collectively, our results indicate that SIM inhibits cell proliferation and arrests NCI-H460 cell cycle progression via inhibition of cyclin-dependent kinases and cyclins and the enhancement of CDK inhibitors p16 and p27. Our findings suggest that, in addition to the known effects on hypercholesterolemia therapy, SIM may also provide antitumor activity in established NSCLC. PMID:23481641

  3. Juvenile hormone biosynthesis in adult Blattella germanica requires nuclear receptors Seven-up and FTZ-F1

    PubMed Central

    Borras-Castells, Ferran; Nieva, Claudia; Maestro, José L.; Maestro, Oscar; Belles, Xavier; Martín, David

    2017-01-01

    In insects, the transition from juvenile development to the adult stage is controlled by juvenile hormone (JH) synthesized from the corpora allata (CA) glands. Whereas a JH-free period during the last juvenile instar triggers metamorphosis and the end of the growth period, the reappearance of this hormone after the imaginal molt marks the onset of reproductive adulthood. Despite the importance of such transition, the regulatory mechanism that controls it remains mostly unknown. Here, using the hemimetabolous insect Blattella germanica, we show that nuclear hormone receptors Seven-up-B (BgSvp-B) and Fushi tarazu-factor 1 (BgFTZ-F1) have essential roles in the tissue- and stage-specific activation of adult CA JH-biosynthetic activity. Both factors are highly expressed in adult CA cells. Moreover, RNAi-knockdown of either BgSvp-B or BgFTZ-F1 results in adult animals with a complete block in two critical JH-dependent reproductive processes, vitellogenesis and oogenesis. We show that this reproductive blockage is the result of a dramatic impairment of JH biosynthesis, due to the CA-specific reduction in the expression of two key JH biosynthetic enzymes, 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 (BgHMG-S1) and HMG-reductase (BgHMG-R). Our findings provide insights into the regulatory mechanisms underlying the specific changes in the CA gland necessary for the proper transition to adulthood. PMID:28074850

  4. Effects of gibberellic acid on primary terpenoids and delta-tetrahydrocannabinol in Cannabis sativa at flowering stage.

    PubMed

    Mansouri, Hakimeh; Asrar, Zahra; Mehrabani, Mitra

    2009-06-01

    Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA(3)) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA(3) caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Delta(9)-tetrahydrocannabinol (THC) contents and an increase in alpha-tocopherol content. The treated plants with GA(3) showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and beta-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA(3) can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.

  5. 27-Hydroxycholesterol contributes to disruptive effects on learning and memory by modulating cholesterol metabolism in the rat brain.

    PubMed

    Zhang, D-D; Yu, H-L; Ma, W-W; Liu, Q-R; Han, J; Wang, H; Xiao, R

    2015-08-06

    Cholesterol metabolism is important for neuronal function in the central nervous system (CNS). The oxysterol 27-hydroxycholesterol (27-OHC) is a cholesterol metabolite that crosses the blood-brain barrier (BBB) and may be a useful substitutive marker for neurodegenerative diseases. However, the effects of 27-OHC on learning and memory and the underlying mechanisms are unclear. To determine this mechanism, we investigated learning and memory and cholesterol metabolism in rat brain following the injection of various doses of 27-OHC into the caudal vein. We found that 27-OHC increased cholesterol levels and upregulated the expression of liver X receptor-α (LXR-α) and adenosine triphosphate (ATP)-binding cassette transporter protein family member A1 (ABCA1). In addition, 27-OHC decreased the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR) and low-density lipoprotein receptor (LDLR) in rat brain tissues. These findings suggest that 27-OHC may negatively modulate cognitive effects and cholesterol metabolism in the brain.

  6. Optimal Low-Density Lipoprotein Cholesterol for Cardiovascular Prevention: How Low Should We Go?

    PubMed

    Anderson, Todd J

    2017-03-01

    The treatment of dyslipidemia with lifestyle interventions and statin-based therapy has been an important defense against atherosclerotic cardiovascular disease and its complications. It has been well documented for more than 2 decades that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) reduce the risk of events. The evolution of drug development and randomized clinical trials in cardiovascular medicine has resulted in the conclusion that lower cholesterol concentrations result in greater benefit. However, how aggressive one should be in lowering cholesterol levels and to what level has not been definitively established. In this brief review I aim to defend the hypothesis that lower is better on the basis of the evidence to date. This will include indirect evidence from randomized clinical trials with statins and novel lipid-modifying drugs. In addition, there is a wealth of epidemiology and Mendelian randomization genetic data to support this. Also, on-treatment low-density lipoprotein cholesterol concentrations show a robust relationship with cardiovascular disease events. Finally, most national guidelines groups around the world continue to advocate for a treat to target philosophy. As such, the prevailing philosophy is that lowering low-density lipoprotein cholesterol to very low levels is our best preventative strategy particularly for those at the highest risk. We eagerly await the results of ongoing clinical trials that will more firmly establish if this concept will ultimately be proven correct.

  7. A call for the better utilization of physical activity and exercise training in the defense against cardiovascular disease.

    PubMed

    Murlasits, Zsolt

    2015-11-01

    Statins, also known as 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, effectively reduce elevated levels of serum LDL-C concentration and in turn lower cardiovascular morbidity and mortality. Regular exercise and physical activity also have significant preventive effects against cardiovascular diseases by simultaneously reducing multiple risk factors. However, statins also produce a number of adverse events, including muscle pain, which increases dramatically in statin users who also exercise, likely limiting the cardiovascular benefits. Most importantly, reduced physical activity participation due to statin-related side effects can cancel out the benefits of the pharmacological treatment. Although exercise training offers more modest benefits compared to pharmacological therapy against traditional risk factors, considering the total impact of exercise on cardiovascular health, it is now evident that this intervention may offer a greater reduction of risks compared to statin therapy alone. However, primary recommendations regarding cardiovascular therapy still center around pharmacological approaches. Thus a new outlook is called for in clinical practice that provides room for physical activity and exercise training, thus lipid targets can be reached by a combined intervention along with improvements in other cardiovascular parameters, such as endothelial function and low-grade inflammation. Databases such as Pubmed and Google Scholar as well as the reference list of the relevant articles were searched to collect information for this opinion article.

  8. Protective role of puerarin on lead-induced alterations of the hepatic glutathione antioxidant system and hyperlipidemia in rats.

    PubMed

    Liu, Chan-Min; Ma, Jie-Qiong; Sun, Yun-Zhi

    2011-12-01

    Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. The aim of the present study was to investigate the effects of puerarin on hepatic oxidative stress and hyperlipidemia in rats exposed to lead. Our data showed that puerarin significantly prevented lead-induced hepatotoxicity, indicated by both diagnostic indicators of liver damage (serum aminotransferase levels) and histopathological analysis. Moreover, lead-induced profound elevation of ROS production and oxidative stress, as evidenced by increasing of lipid peroxidation level, reducing of GPx, GST, GR and GCL activities and depleting of intracellular reduced GSH level in liver, were suppressed by treatment with puerarin. Furthermore, the increase of serum cholesterol, triglycerides and LDL induced by lead was effectively suppressed by puerarin. The HDL level in the lead treatment rats was also increased by puerarin. Western blot analysis showed that puerarin remarkably inhibited hyperlipidemia by regulating the expression of cholesterol 7a-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and low-density lipoprotein receptor (LDL-R) in liver of lead treated rats. Altogether, these results suggest that puerarin could protect the lead-induced liver injury and hyperlipidemia by reducing ROS production, renewing the activities of antioxidant enzymes and influencing expression of hepatic lipid biosynthesis and metabolism genes.

  9. Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD.

    PubMed

    Famer, D; Wahlund, L-O; Crisby, M

    2010-11-12

    We have previously shown that a high cholesterol (HC) diet results in increases in microglia load and levels of the pro-inflammatory cytokine interleukin-6 (IL-6) in the brains of wild type (WT) and apolipoprotein E knockout (ApoE-/-) mice. In the present investigation, we analyzed whether treatment with rosuvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, would prevent the increases in inflammatory microglia and IL-6 levels in the brain and plasma of WT and ApoE-/- mice. We report that a HC diet resulted in an increased microglia load in the brains of WT and ApoE-/- mice, in support of our previous study. Treatment with rosuvastatin significantly decreased the microglia load in the brains of WT and ApoE-/- mice on a HC diet. Rosuvastatin treatment resulted in lowered plasma IL-6 levels in WT mice on a HC diet. However, in the present study the number of IL-6 positive cells in the brain was not significantly affected by a HC diet. A recent clinical study has shown that rosuvastatin reduces risk of ischemic stroke in patients with high plasma levels of the inflammatory marker C-reactive protein by 50%. The results from our study show that rosuvastatin reduces inflammatory cells in the brain. This finding is essential for furthering the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and stroke.

  10. Identification of transformation products of rosuvastatin in water during ZnO photocatalytic degradation through the use of associated LC-QTOF-MS to computational chemistry.

    PubMed

    Segalin, Jéferson; Sirtori, Carla; Jank, Louíse; Lima, Martha F S; Livotto, Paolo R; Machado, Tiele C; Lansarin, Marla A; Pizzolato, Tânia M

    2015-12-15

    Rosuvastatin (RST), a synthetic statin, is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, with a number of pleiotropic properties, such as anti-inflammation, antioxidation and cardiac remodelling attenuation. According to IMS Health, rosuvastatin was the third best-selling drug in the United States in 2012. RST was recently found in European effluent samples at a detection frequency of 36%. In this study, we evaluate the identification process of major transformation products (TPs) of RST generated during the heterogeneous photocatalysis process with ZnO. The degradation of the parent molecule and the identification of the main TPs were studied in demineralised water. The TPs were monitored and identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS). Ten TPs were tentatively identified and some of them originated from the hydroxylation suffered by the aromatic ring during the initial stages of the process. Structural elucidation of some of the most abundant or persistent TPs was evaluated by computational analysis, which demonstrated that this approach can be used as a tool to help the elucidation of structures of unknown molecules. The analysis of the parameters obtained from ab initio calculations for different isomers showed the most stable structures and, consequently, the most likely to be found.

  11. Fruiting body of Niuchangchih (Antrodia camphorata) protects livers against chronic alcohol consumption damage.

    PubMed

    Huang, Chia-Hsin; Chang, Yuan-Yen; Liu, Cheng-Wei; Kang, Wen-Yu; Lin, Yi-Ling; Chang, Hsien-Chang; Chen, Yi-Chen

    2010-03-24

    An alcoholic fatty liver disease was induced by drinking water containing 20% (w/w) alcohol. Therapeutic groups were orally administrated dosages of 0.25 g silymarin/kg body weight (BW) and a low dosage of Niuchangchih (Antrodia camphorata) (0.025 g/kg BW) and a high dosage of Niuchangchih (0.1 g/kg BW) per day. Niuchangchih, especially at the high dosage, not only showed a hypercholesterolemic effect (p < 0.05) but also reduced (p < 0.05) hepatic lipids in alcohol-fed rats. Those beneficial effects could be partially attributed to higher (p < 0.05) fecal cholesterol and bile acid outputs, as well as downregulations (p < 0.05) of 3-hydroxy-3-methylglutaryl-CoA reductase, sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme gene expressions; meanwhile, there was an upregulation of low-density lipoprotein receptor and peroxisome proliferator-activated alpha gene expression. Besides, Niuchangchih also enhanced (p < 0.05) the liver glutathione, Trolox equivalent antioxidant capacity, and activities of superoxide dismutase, catalase, and glutathione peroxidase and decreased the liver malondialdehyde content, which also partially contributed to the lowered (p < 0.05) serum aspartate aminotransferase levels and no observed lesion in the histological examination of alcohol-fed rats.

  12. Statins May Reduce Breast Cancer Risk, Particularly Hormone Receptor-Negative Disease.

    PubMed

    Vinayak, Shaveta; Kurian, Allison W

    2009-09-01

    Estrogen and progesterone receptor-negative breast cancer disproportionately affects young women and African Americans, has a poor prognosis, and lacks an effective chemoprevention agent. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, known as "statins," are appealing candidate agents for breast cancer chemoprevention because of their demonstrated safety after decades of widespread use. In preclinical studies, statins inhibit multiple cancer-associated pathways in both hormone receptor (HR)-negative and HR-positive cell lines. Epidemiologic studies of statins and breast cancer show inconsistent results, with some suggesting a reduction in HR-negative breast cancer incidence in lipophilic statin users. However, large meta-analyses show no association between statin use and overall risk of breast cancer, although most did not evaluate tumor HR status. Multiple phase 1 and 2 prevention studies of statins for breast cancer risk reduction are ongoing. If results are promising, they may justify a randomized trial of statins for breast cancer chemoprevention, with a focus on HR-negative disease.

  13. The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change*

    PubMed Central

    Howe, Vicky; Chua, Ngee Kiat; Stevenson, Julian; Brown, Andrew J.

    2015-01-01

    Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide. We have identified a region integrally associated with the endoplasmic reticulum membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system because degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis. PMID:26434806

  14. Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation

    PubMed Central

    Torabi, Sheida

    2015-01-01

    Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25–75 μmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25–75 μmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake. PMID:26660152

  15. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    SciTech Connect

    Wada, Hiromichi Abe, Mitsuru; Ono, Koh; Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide; Satoh, Noriko; Fujita, Masatoshi; Kita, Toru; Shimatsu, Akira; Hasegawa, Koji

    2008-10-03

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 {mu}M of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-{alpha}-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 {mu}M), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

  16. Pharmacy benefits management in the Veterans Health Administration: 1995 to 2003.

    PubMed

    Sales, Mariscelle M; Cunningham, Francesca E; Glassman, Peter A; Valentino, Michael A; Good, Chester B

    2005-02-01

    The Department of Veterans Affairs (VA) Pharmacy Benefits Management Strategic Healthcare Group (VA PBM) oversees the formulary for the entire VA system, which serves more than 4 million veterans and provides more than 108 million prescriptions per year. Since its establishment in 1995, the VA PBM has managed pharmaceuticals and pharmaceutical-related policies, including drug safety and efficacy evaluations, pharmacologic management algorithms, and criteria for drug use. These evidence-based practices promote, optimize, and assist VA providers with the safe and appropriate use of pharmaceuticals while allowing for formulary decisions that can result in substantial cost savings. The VA PBM also has utilized various contracting techniques to standardize generic agents as well as specific drugs and drug classes (eg, antihistamines, angiotensin-converting enzyme inhibitors, alpha-blockers, and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors [statins]). These methods have enabled the VA to save approximately dollar 1.5 billion since 1996 even as drug expenditures continued to rise from roughly dollar 1 billion in fiscal year (FY) 1996 to more than dollar 3 billion in FY 2003. Furthermore, the VA PBM has established an outcomes research section to undertake quality-improvement and safety initiatives that ultimately monitor and determine the clinical impact of formulary decisions on the VA system nationwide. The experiences of this pharmacy benefits program, including clinical and contracting processes/procedures and their impact on the VA healthcare system, are described.

  17. Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis.

    PubMed

    Healey, Gareth D; Collier, Christine; Griffin, Sholeem; Schuberth, Hans-Joachim; Sandra, Olivier; Smith, David G; Mahan, Suman; Dieuzy-Labaye, Isabelle; Sheldon, I Martin

    2016-01-15

    Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies.

  18. Mevalonate Biosynthesis Intermediates Are Key Regulators of Innate Immunity in Bovine Endometritis

    PubMed Central

    Collier, Christine; Griffin, Sholeem; Schuberth, Hans-Joachim; Sandra, Olivier; Smith, David G.; Mahan, Suman; Dieuzy-Labaye, Isabelle; Sheldon, I. Martin

    2016-01-01

    Metabolic changes can influence inflammatory responses to bacteria. To examine whether localized manipulation of the mevalonate pathway impacts innate immunity, we exploited a unique mucosal disease model, endometritis, where inflammation is a consequence of innate immunity. IL responses to pathogenic bacteria and LPS were modulated in bovine endometrial cell and organ cultures by small molecules that target the mevalonate pathway. Treatment with multiple statins, bisphosphonates, squalene synthase inhibitors, and small interfering RNA showed that inhibition of farnesyl-diphosphate farnesyl transferase (squalene synthase), but not 3-hydroxy-3-methylglutaryl-CoA reductase or farnesyl diphosphate synthase, reduced endometrial organ and cellular inflammatory responses to pathogenic bacteria and LPS. Although manipulation of the mevalonate pathway reduced cellular cholesterol, impacts on inflammation were independent of cholesterol concentration as cholesterol depletion using cyclodextrins did not alter inflammatory responses. Treatment with the isoprenoid mevalonate pathway-intermediates, farnesyl diphosphate and geranylgeranyl diphosphate, also reduced endometrial cellular inflammatory responses to LPS. These data imply that manipulating the mevalonate pathway regulates innate immunity within the endometrium, and that isoprenoids are regulatory molecules in this process, knowledge that could be exploited for novel therapeutic strategies. PMID:26673142

  19. Long-term safety and efficacy profile of simvastatin.

    PubMed

    Boccuzzi, S J; Bocanegra, T S; Walker, J F; Shapiro, D R; Keegan, M E

    1991-11-01

    Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, has been administered to approximately 2,400 patients with primary hypercholesterolemia with a mean follow-up of 1 year in controlled clinical studies and their open extensions. Approximately 10% of this population received simvastatin for a period of greater than or equal to 2 years. The population on whom this safety analysis is based had a mean age of 50 years; 62% were men and approximately 27% had preexisting coronary artery disease. Simvastatin was titrated to the maximal daily dose of 40 mg each evening in 56% of the study population (last recorded dose). The most frequently reported drug-related clinical adverse experiences were constipation (2.5%), abdominal pain (2.2%), flatulence (2.0%) and headaches (1%). Persistent elevations of serum transaminase levels greater than 3 times the upper limit of normal were observed in only 1% of this cohort with only 0.1% of the total population requiring discontinuation of therapy. There were no clinically apparent episodes of hepatitis. Discontinuation of therapy due to myopathy was extremely rare (0.08%). Only minimal increases in the frequency of lens opacities (1%) were observed from baseline to the last lens examination during follow-up, consistent with the expected increase in lens opacity development due to normal aging. Patients who were greater than or equal to 65 years old had a clinical and laboratory safety profile comparable to the nonelderly population.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Simvastatin treatment highlights a new role for the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and cognitive performance in rats.

    PubMed

    Segatto, Marco; Manduca, Antonia; Lecis, Claudio; Rosso, Pamela; Jozwiak, Adam; Swiezewska, Ewa; Moreno, Sandra; Trezza, Viviana; Pallottini, Valentina

    2014-03-01

    The aim of the present work was to shed light on the role played by the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and memory consolidation in rodents through the inhibition of the key and rate-limiting enzyme 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR) both in vivo and in vitro with simvastatin. Three-month-old male Wistar rats treated for 21 days with simvastatin or vehicle were tested in the social interaction, elevated plus-maze, and inhibitory avoidance tasks; after behavioral testing, the amygdala, hippocampus, prefrontal cortex, dorsal, and ventral striatum were dissected out for biochemical assays. In order to delve deeper into the molecular mechanisms underlying the observed effects, primary rat hippocampal neurons were used. Our results show that HMGR inhibition by simvastatin induces anxiogenic-like effects in the social interaction but not in the elevated plus-maze test, and improves memory consolidation in the inhibitory avoidance task. These effects are accompanied by imbalances in the activity of specific prenylated proteins, Rab3 and RhoA, involved in neurotransmitter release, and synaptic plasticity, respectively. Taken together, the present findings indicate that the isoprenoid/cholesterol biosynthetic pathway is critically involved in the physiological modulation of both emotional and cognitive processes in rodents.

  1. IL-1 regulates the Cyp7a1 gene and serum total cholesterol level at steady state in mice.

    PubMed

    Kojima, Misaki; Ashino, Takashi; Yoshida, Takemi; Iwakura, Yoichiro; Sekimoto, Masashi; Degawa, Masakuni

    2009-02-06

    We examined the role of hepatic interleukin (IL)-1alpha/beta in serum total cholesterol homeostasis using male and female IL-1-knockout (KO) mice and wild-type (WT) mice. Serum total cholesterol level was higher in males than in females in WT and KO mice. The difference between sexes was closely correlated with the difference in gene expression level of cholesterol 7alpha-hydroxylase (Cyp7a1), a rate-limiting enzyme for bile acid synthesis. No significant sex difference in gene expression level of 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-limiting enzyme for cholesterol synthesis, was observed in WT mice. Interestingly, the gene expression level of hepatic Cyp7a1 was lower in KO mice than in sex-matched WT mice, while the serum total cholesterol level was the opposite. The present findings demonstrate that IL-1alpha and IL-1beta are positive regulators for the Cyp7a1 gene in steady-state mice and that Cyp7a1 is one of the factors that mediate the difference in serum total cholesterol level between sexes.

  2. Hypocholesterolemic activity of grape seed proanthocyanidin is mediated by enhancement of bile acid excretion and up-regulation of CYP7A1.

    PubMed

    Jiao, Rui; Zhang, Zesheng; Yu, Hongjian; Huang, Yu; Chen, Zhen-Yu

    2010-11-01

    Interest in grape seed proanthocyanidin (GSP) as a cholesterol-lowering nutraceutical is growing. This study was to investigate the effect of GSP on blood cholesterol level and gene expression of cholesterol-regulating enzymes in Golden Syrian hamsters maintained on a 0.1% cholesterol diet. Results affirmed supplementation of 0.5% or 1.0% GSP could decrease plasma total cholesterol and triacylglycerol level. Western blot and real-time polymerase chain reaction analyses demonstrated GSP did not affect sterol regulatory element binding protein-2 and low-density lipoprotein receptor; however, it increased mRNA 3-hydroxy-3-methylglutaryl coenzyme A reductase. GSP had no effect on the protein mass of liver X receptor alpha (LXRα) but it decreased mRNA LXRα. Most importantly, GSP increased not only the protein level of cholesterol-7α-hydroxylase (CYP7A1) but also mRNA CYP7A1. It was concluded that the hypocholesterolemic activity of GSP was most likely mediated by enhancement of bile acid excretion and up-regulation of CYP7A1.

  3. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking

    PubMed Central

    Islam, Barira; Sharma, Charu; Adem, Abdu; Aburawi, Elhadi; Ojha, Shreesh

    2015-01-01

    Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP+). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (–)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure–function relationship studies. PMID:26357462

  4. Endothelial dysfunction, oxidative stress and inflammation in atherosclerosis: beneficial effects of statins.

    PubMed

    Lahera, Vicente; Goicoechea, Marian; de Vinuesa, Soledad García; Miana, María; de las Heras, Natalia; Cachofeiro, Victoria; Luño, José

    2007-01-01

    Atherosclerosis and its complications represent the major cause of death in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A [HMGCoA] reductase and consequently inhibitors of cholesterol biosynthesis. Statins have been described as the most potent class of drugs to reduce serum cholesterol levels. In clinical trials, statins are beneficial in primary and secondary prevention of coronary heart disease. Statins, were initially designed as cholesterol-lowering drugs. However, these drugs, besides their lipid-lowering properties, exert a number of protective effects on the cardiovascular system that emerged over the past years. The benefits observed with statin treatment appear to be greater than that might be expected from reduction in lipid levels alone, suggesting effects beyond cholesterol lowering. These cholesterol-independent effects have been called "pleiotropic". The cholesterol-independent or "pleiotropic" effects of statins involve improvement of endothelial function, stability of atherosclerotic plaques, decrease of oxidative stress and inflammation, and inhibition of thrombogenic response. These pleiotropic effects of statins have been proposed as key properties of these drugs to reduce cardiovascular morbidity and mortality. The present review will emphasize the molecular mechanisms underlying the effects of statins on endothelial function and oxidative stress. In particular, inhibition of small GTP-binding proteins, Rho, Ras and Rac, which are regulated by isoprenoids [farnesyl pyrophosphate and geranylgeranyl pyrophosphate], seems to play an important role in mediating the pleiotropic effects of statins.

  5. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

    PubMed

    Hemmerlin, Andréa; Hoeffler, Jean-François; Meyer, Odile; Tritsch, Denis; Kagan, Isabelle A; Grosdemange-Billiard, Catherine; Rohmer, Michel; Bach, Thomas J

    2003-07-18

    In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

  6. Involvement of membrane sterols in hypergravity-induced modifications of growth and cell wall metabolism in plant stems

    NASA Astrophysics Data System (ADS)

    Koizumi, T.; Soga, K.; Wakabayashi, K.; Suzuki, M.; Muranaka, T.; Hoson, T.

    Organisms living on land resist the gravitational force by constructing a tough body Plants have developed gravity resistance responses after having first went ashore more than 500 million years ago The mechanisms of gravity resistance responses have been studied under hypergravity conditions which are easily produced on earth by centrifugation In Arabidopsis hypocotyls hypergravity treatment greatly increased the expression level of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase HMGR which is involved in synthesis of terpenoids such as membrane sterols In the present study we examined the role of membrane sterols in gravity resistance in plants by analyzing sterol levels of stem organs grown under hypergravity conditions and by analyzing responses to hypergravity of the organs whose sterol level was modulated Hypergravity inhibited elongation growth but stimulated lateral expansion of Arabidopsis hypocotyls and azuki bean epicotyls Under hypergravity conditions sterol levels were kept high as compared with 1 g controls during incubation Lovastatin an inhibitor HMGR prevented lateral expansion as the gravity resistance response in azuki bean epicotyls Similar results were obtained in analyses with loss of function mutants of HMGR in Arabidopsis It has been shown that sterols play a role in cellulose biosynthesis probably as the primer In wild type Arabidopsis hypocotyls hypergravity increased the cellulose content but it did not influence the content in HMGR mutants These results suggest that hypergravity increases

  7. [Autoantibodies of Inflammatory Myopathies: Update].

    PubMed

    Suzuki, Shigeaki

    2016-12-01

    Inflammatory myopathies are a heterogeneous group of immune-mediated diseases that involve the skeletal muscle as well as many other organs. In addition to a histological diagnosis at muscle biopsy, the clinical phenotypes of inflammatory myopathies can be defined by the presence of various autoantibodies that are originally detected by RNA or protein immunoprecipitation. However, the correlation between histological features and autoantibodies has not been fully elucidated. Immune-mediated necrotizing myopathy (IMNM), which is characterized by significant necrotic and regeneration muscle fibers with minimal or no inflammatory cell infiltration, is associated with the presence of autoantibodies. IMNM is now classified as a distinct category of inflammatory myopathies, separate from polymyositis, dermatomyositis, and sporadic inclusion body myositis. Here, we divided the autoantibodies of inflammatory myopathies into the following categories: those associated with IMNM, those with activity against aminoacyl transfer RNA synthetase, those associated with dermatomyositis, and those related to other disorders, including overlap syndrome, inclusion body myositis, and primary biliary cirrhosis. The detection of autoantibodies against signal recognition particle or 3-hydroxy-3-methylglutaryl-coenzyme A reductase is useful for the diagnosis of IMNM. The screening of autoantibodies has clinical relevance for managing patients with inflammatory myopathies.

  8. [Statins and mitochondria].

    PubMed

    Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

    2016-01-01

    The aim of this review is to report on influence of statins on mitochondria function. Statins are serum cholesterol-lowering drugs. They act by competitively inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), the first committed enzyme of the mevalonate pathway. In this way, statins inhibit the endogenous cholesterol synthesis. Emerging evidence suggest that statins impair mitochondria, which is demonstrated by abnormal mitochondrial morphology, decreased oxidative phosphorylation capacity and yield, decreased mitochondrial membrane potential and activation of intrinsic apoptotic pathway. Mechanisms of statin-induced mitochondrial dysfunction are not fully understood. The following causes are proposed: (i) deficiency of coenzyme Q10, an important electron carrier of mitochondrial respiratory chain; (ii) inhibition of respiratory chain complexes; (iii) inhibitory effect on protein prenylation; and (iv) induction of mitochondrial apoptosis pathway. These phenomena could play a significant role in the etiology of statin-induced disease, especially myopathy. Studies on statin-induced mitochondrial apoptosis could be useful in developing a new cancer therapy.

  9. The molecular mechanism of the cholesterol-lowering effect of dill and kale: The influence of the food matrix components.

    PubMed

    Danesi, Francesca; Govoni, Marco; D'Antuono, Luigi Filippo; Bordoni, Alessandra

    2016-07-01

    Foods are complex matrices containing many different compounds, all of which contribute to the overall effect of the food itself, although they have different mechanisms of action. While evaluating the effect of bioactive compounds, it is important to consider that the use of a single compound can hide the effects of the other molecules that can act synergistically or antagonistically in the same food. The aim of the present study was to evaluate the influence of food matrix components by comparing two edible plants (dill and kale) with cholesterol-lowering potential and similar contents of their most representative bioactive, quercetin. The molecular effects of the extracts were evaluated in HepG2 cells by measuring the expression of sterol-regulatory element-binding proteins (SREBPs), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and low density lipoprotein receptor (LDLR) at the mRNA and protein level. The results reported here show that both extracts reduced the cellular cholesterol level with a similar trend and magnitude. It is conceivable that the slightly different results are due to the diverse composition of minor bioactive compounds, indicating that only by considering food as a whole is it possible to understand the complex relationship between food, nutrition, and health in a foodomics vision.

  10. Algal sterols are as effective as β-sitosterol in reducing plasma cholesterol concentration.

    PubMed

    Chen, Jingnan; Jiao, Rui; Jiang, Yue; Bi, Yanlan; Chen, Zhen-Yu

    2014-01-22

    The present study examined the cholesterol-lowering activity of sterol extract (SE) derived from alga Schizochytrium sp. and its interaction with gene expression of transporters, receptors, and enzymes involved in cholesterol absorption and metabolism. GC-MS analyses found that SE was a mixture of various sterols including lathosterol, ergosterol, stigmasterol, 24-ethylcholesta-5,7,22-trienol, stigmasta-7,24(24(1))-dien-3β-ol, and cholesterol. Results showed that SE at doses of 0.06 and 0.30 g/kg diet were able to decrease plasma cholesterol concentration by 19.5 and 34%, respectively, compared with the control, in hamsters maintained on a 0.1% high-cholesterol diet. SE at a dose of 0.30 g/kg diet was as effective as β-sitosterol in reducing plasma total cholesterol (TC). SE-induced reduction in plasma TC was accompanied by down-regulation of intestinal acyl-CoA:cholesterol acyltransferase 2 (ACAT2) and hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and up-regulation of hepatic low-density lipoprotein (LDL) receptor. Addition of SE to the diet increased the excretion of total fecal sterols. It was concluded that SE possessed the same cholesterol-lowering activity as β-sitosterol and the underlying mechanisms were mediated by increasing sterol excretion and decreasing cholesterol absorption and synthesis.

  11. Juvenile hormone biosynthesis in adult Blattella germanica requires nuclear receptors Seven-up and FTZ-F1.

    PubMed

    Borras-Castells, Ferran; Nieva, Claudia; Maestro, José L; Maestro, Oscar; Belles, Xavier; Martín, David

    2017-01-11

    In insects, the transition from juvenile development to the adult stage is controlled by juvenile hormone (JH) synthesized from the corpora allata (CA) glands. Whereas a JH-free period during the last juvenile instar triggers metamorphosis and the end of the growth period, the reappearance of this hormone after the imaginal molt marks the onset of reproductive adulthood. Despite the importance of such transition, the regulatory mechanism that controls it remains mostly unknown. Here, using the hemimetabolous insect Blattella germanica, we show that nuclear hormone receptors Seven-up-B (BgSvp-B) and Fushi tarazu-factor 1 (BgFTZ-F1) have essential roles in the tissue- and stage-specific activation of adult CA JH-biosynthetic activity. Both factors are highly expressed in adult CA cells. Moreover, RNAi-knockdown of either BgSvp-B or BgFTZ-F1 results in adult animals with a complete block in two critical JH-dependent reproductive processes, vitellogenesis and oogenesis. We show that this reproductive blockage is the result of a dramatic impairment of JH biosynthesis, due to the CA-specific reduction in the expression of two key JH biosynthetic enzymes, 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 (BgHMG-S1) and HMG-reductase (BgHMG-R). Our findings provide insights into the regulatory mechanisms underlying the specific changes in the CA gland necessary for the proper transition to adulthood.

  12. Potato steroidal glycoalkaloid levels and the expression of key isoprenoid metabolic genes.

    PubMed

    Krits, Pinchas; Fogelman, Edna; Ginzberg, Idit

    2007-12-01

    The potato steroidal glycoalkaloids (SGA) are toxic secondary metabolites, and their total content in tubers should not exceed 20 mg/100 g fresh weight. The two major SGA in cultivated potato (Solanum tuberosum) are alpha-chaconine and alpha-solanine. SGA biosynthetic genes and the genetic factors that control their expression have not yet been determined. In the present study, potato genotypes exhibiting different levels of SGA content showed an association between high SGA levels in their leaves and tubers and high expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (hmg1) and squalene synthase 1 (pss1), genes of the mevalonic/isoprenoid pathway. Transcripts of other key enzymes of branches of the isoprenoid pathway, vetispiradiene/sesquiterpene synthase (pvs1) and sterol C24-methyltransferase type1 (smt1), were undetectable or exhibited stable expression regardless of SGA content, respectively, suggesting facilitated precursor flow to the SGA biosynthetic branch. The transcript ratio of solanidine glucosyltransferase (sgt2) to solanidine galactosyltransferase (sgt1) was correlated to the documented chaconine-to-solanine ratio in the tested genotypes. Significantly higher expression of hmg1, pss1, smt1, sgt1 and sgt2 was monitored in the tuber phelloderm than in the parenchyma of the tuber's flesh, targeting the former as the main SGA-producing tissue in the tuber, in agreement with the known high SGA content in the layers directly under the tuber skin.

  13. Inhibitory Effect of Statins on Inflammation-Related Pathways in Human Abdominal Aortic Aneurysm Tissue

    PubMed Central

    Yoshimura, Koichi; Nagasawa, Ayako; Kudo, Junichi; Onoda, Masahiko; Morikage, Noriyasu; Furutani, Akira; Aoki, Hiroki; Hamano, Kimikazu

    2015-01-01

    HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors (statins) have been suggested to attenuate abdominal aortic aneurysm (AAA) growth. However, the effects of statins in human AAA tissues are not fully elucidated. The aim of this study was to investigate the direct effects of statins on proinflammatory molecules in human AAA walls in ex vivo culture. Simvastatin strongly inhibited the activation of nuclear factor (NF)-κB induced by tumor necrosis factor (TNF)-α in human AAA walls, but showed little effect on c-jun N-terminal kinase (JNK) activation. Simvastatin, as well as pitavastatin significantly reduced the secretion of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-2 and epithelial neutrophil-activating peptide (CXCL5) under both basal and TNF-α-stimulated conditions. Similar to statins, the Rac1 inhibitor NSC23766 significantly inhibited the activation of NF-κB, accompanied by a decreased secretion of MMP-9, MCP-2 and CXCL5. Moreover, the effect of simvastatin and the JNK inhibitor SP600125 was additive in inhibiting the secretion of MMP-9, MCP-2 and CXCL5. These findings indicate that statins preferentially inhibit the Rac1/NF-κB pathway to suppress MMP-9 and chemokine secretion in human AAA, suggesting a mechanism for the potential effect of statins in attenuating AAA progression. PMID:25993292

  14. Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress.

    PubMed

    Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M M; Pandey, Rakesh

    2017-02-03

    Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress.

  15. Lipidomic-based investigation into the regulatory effect of Schisandrin B on palmitic acid level in non-alcoholic steatotic livers

    PubMed Central

    Kwan, Hiu Yee; Niu, Xuyan; Dai, Wenlin; Tong, Tiejun; Chao, Xiaojuan; Su, Tao; Chan, Chi Leung; Lee, Kim Chung; Fu, Xiuqiong; Yi, Hua; Yu, Hua; Li, Ting; Tse, Anfernee Kai Wing; Fong, Wang Fun; Pan, Si-Yuan; Lu, Aiping; Yu, Zhi-Ling

    2015-01-01

    Schisandrin B (SchB) is one of the most abundant bioactive dibenzocyclooctadiene derivatives found in the fruit of Schisandra chinensis. Here, we investigated the potential therapeutic effects of SchB on non-alcoholic fatty-liver disease (NAFLD). In lipidomic study, ingenuity pathway analysis highlighted palmitate biosynthesis metabolic pathway in the liver samples of SchB-treated high-fat-diet-fed mice. Further experiments showed that the SchB treatment reduced expression and activity of fatty acid synthase, expressions of hepatic mature sterol regulatory element binding protein-1 and tumor necrosis factor-α, and hepatic level of palmitic acid which is known to promote progression of steatosis to steatohepatitis. Furthermore, the treatment also reduced hepatic fibrosis, activated nuclear factor-erythroid-2-related factor-2 which is known to attenuate the progression of NASH-related fibrosis. Interestingly, in fasting mice, a single high-dose SchB induced transient lipolysis and increased the expressions of adipose triglyceride lipase and phospho-hormone sensitive lipase. The treatment also increased plasma cholesterol levels and 3-hydroxy-3-methylglutaryl-CoA reductase activity, reduced the hepatic low-density-lipoprotein receptor expression in these mice. Our data not only suggest SchB is a potential therapeutic agent for NAFLD, but also provided important information for a safe consumption of SchB because SchB overdosed under fasting condition will have adverse effects on lipid metabolism. PMID:25766252

  16. Elicitation of Diosgenin Production in Trigonella foenum-graecum (Fenugreek) Seedlings by Methyl Jasmonate.

    PubMed

    Chaudhary, Spandan; Chikara, Surendra K; Sharma, Mahesh C; Chaudhary, Abhinav; Alam Syed, Bakhtiyar; Chaudhary, Pooja S; Mehta, Aditya; Patel, Maulik; Ghosh, Arpita; Iriti, Marcello

    2015-12-15

    The effects of methyl jasmonate (MeJA), an elicitor of plant defense mechanisms, on the biosynthesis of diosgenin, a steroidal saponin, were investigated in six fenugreek (Trigonella foenum-graecum) varieties (Gujarat Methi-2, Kasuri-1, Kasuri-2, Pusa Early Branching, Rajasthan Methi and Maharashtra Methi-5). Treatment with 0.01% MeJA increased diosgenin levels, in 12 days old seedlings, from 0.5%-0.9% to 1.1%-1.8%. In addition, MeJA upregulated the expression of two pivotal genes of the mevalonate pathway, the metabolic route leading to diosgenin: 3-hydroxy-3-methylglutaryl-CoA reductase (HMG) and sterol-3-β-glucosyl transferase (STRL). In particular, MeJA increased the expression of HMG and STRL genes by 3.2- and 22.2-fold, respectively, in the Gujarat Methi-2 variety, and by 25.4- and 28.4-fold, respectively, in the Kasuri-2 variety. Therefore, MeJA may be considered a promising elicitor for diosgenin production by fenugreek plants.

  17. Lovastatin prevents bleomycin-induced DNA damage to HepG2 cells

    PubMed Central

    Nasiri, Marjan; Etebari, Mahmoud; Jafarian-Dehkordi, Abbas; Moradi, Shahla

    2016-01-01

    Lovastatin as a member of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors is used as a lipid-lowering agent. It can also inhibit the formation of hydrogen peroxide and superoxide anion and finally leads to decline in oxidative stress processes. Here, we evaluated whether lovastatin can increase DNA damage resistance of HepG2 cells against genotoxicity of the anticancer drug bleomycin (BLM). HepG2 cells were incubated with different concentrations of lovastatin (0.1, 0.5, 1, 5 µM) before exposure to BLM (0.5 µg/mL for one h). The genotoxic dose of BLM and lovastatin was separately determined and comet assay was used to evaluate the genotoxicity. After trapping cells in agarose coated lames, they were lysed and the electrophoresis was done in alkaline pH, then colored and monitored by florescent microscope. The results of this study indicated that lovastatin in doses lower than 5 µM has genoprotective effect and in doses higher than 50 µM is genotoxic. In conclusion, lovastatin is able to protect genotoxic effects of BLM in HepG2 cells. Further studies are needed to elucidate the mechanism(s) involved in this process. PMID:28003840

  18. Lipid metabolism as a target for brain cancer therapy: synergistic activity of lovastatin and sodium phenylacetate against human glioma cells.

    PubMed

    Prasanna, P; Thibault, A; Liu, L; Samid, D

    1996-02-01

    Malignant gliomas, the most common form of primary brain tumors, are highly dependent on the mevalonate (MVA) pathway for the synthesis of lipid moieties critical to cell replication. Human glioblastoma cells were found to be uniquely vulnerable to growth arrest by lovastatin, a competitive inhibitor of the enzyme regulating MVA synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. The sodium salt of phenylacetic acid (NaPA), an inhibitor of MVA-pyrophosphate decarboxylase, the enzyme that controls MVA use, acted synergistically with lovastatin to suppress malignant growth. When used at pharmacologically attainable concentrations, the two compounds induced profound cytostasis and loss of malignant properties such as invasiveness and expression of the transforming growth factor-beta 2 gene, coding for a potent immunosuppressive cytokine. Supplementation with exogenous ubiquinone, an end product of the MVA pathway, failed to rescue the cells, suggesting that decreased synthesis of intermediary products are responsible for the antitumor effects observed. In addition to blocking the MVA pathway, lovastatin alone and in combination with NaPA increased the expression of the peroxisome proliferator-activated receptor, a transcription factor implicated in the control of lipid metabolism, cell growth, and differentiation. Our results indicate that targeting lipid metabolism with lovastatin, used alone or in combination with the aromatic fatty acid NaPA, may offer a novel approach to the treatment of malignant gliomas.

  19. Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats.

    PubMed

    Ihara, M; Umekawa, H; Takahashi, T; Furuichi, Y

    1998-10-01

    Diets high in linoleic acid (20% safflower oil contained 77.3% linoleic acid, SO-diet) and alpha-linolenic acid (20% perilla oil contained 58.4% alpha-linolenic acid, PO-diet) were fed to rats for 3, 7, 20, and 50 days, and effects of the diets on lipid metabolism were compared. Levels of serum total cholesterol and phospholipids in the rats fed the PO-diet were markedly lower than those fed the SO-diet after the seventh day. In serum and hepatic phosphatidylcholine and phosphatidylethanolamine, the proportion of n-3 fatty acids showed a greater increase in the PO group than in the SO group in the respective feeding-term. At the third and seventh days after the commencement of feeding the experimental diets, expressions of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA were significantly higher in the SO group than those in the PO group, although the difference was not observed in the longer term. There were no significant differences in the LDL receptor mRNA levels between the two groups through the experimental term, except 3-days feeding. These results indicate that alpha-linolenic acid has a more potent serum cholesterol-lowering ability than linoleic acid both in short and long feeding-terms.

  20. The influence of rosuvastatin upon the gastrointestinal microbiota and host gene expression profiles.

    PubMed

    Nolan, James A; Skuse, Peter Henry; Govindarajan, Kalaimathi; Patterson, Elaine; Konstantinidou, Nina; Casey, Patrick G; MacSharry, John; Shanahan, Fergus; Stanton, Catherine; Hill, Colin; Cotter, P D; Joyce, Susan A; Gahan, Cormac Gm

    2017-02-16

    Statins are the most widely prescribed medications worldwide for the treatment of hypercholesterolemia. They inhibit the activity of 3-hydroxy-3-methylglutaryl co enzyme A reductase (HMG-R) an enzyme involved in cholesterol synthesis in higher organisms and in isoprenoid biosynthesis in some bacteria. We hypothesized that statins may influence the microbial community in the gut through either direct inhibition or indirect mechanisms involving alterations to host responses. We therefore examined the impact of rosuvastatin (RSV) on the community structure of the murine gastrointestinal microbiota. RSV was orally administered to mice and the effects upon the gut microbiota, host bile acid profiles and markers of inflammation were analysed. RSV significantly influenced the microbial community in both the caecum and faeces, causing a significant decrease in alpha diversity in the caecum and resulting in a reduction of several physiologically relevant bacterial groups. RSV treatment of mice significantly affected bile acid metabolism and impacted upon expression of inflammatory markers known to influence microbial community structure (including RegIIIγ and Camp) in the gut. This study suggests that a commonly used statin (RSV) leads to an altered gut microbial composition in normal mice with attendant impacts upon local gene expression profiles, a finding which should prompt further studies to investigate the implications of statins for gut microbiota stability and health in humans.

  1. Statin-induced changes in mitochondrial respiration in blood platelets in rats and human with dyslipidemia.

    PubMed

    Vevera, J; Fišar, Z; Nekovářová, T; Vrablík, M; Zlatohlávek, L; Hroudová, J; Singh, N; Raboch, J; Valeš, K

    2016-11-23

    3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used drugs for lowering blood lipid levels and preventing cardiovascular diseases. However, statins can have serious adverse effects, which may be related to development of mitochondrial dysfunctions. The aim of study was to demonstrate the in vivo effect of high and therapeutic doses of statins on mitochondrial respiration in blood platelets. Model approach was used in the study. Simvastatin was administered to rats at a high dose for 4 weeks. Humans were treated with therapeutic doses of rosuvastatin or atorvastatin for 6 weeks. Platelet mitochondrial respiration was measured using high-resolution respirometry. In rats, a significantly lower physiological respiratory rate was found in intact platelets of simvastatin-treated rats compared to controls. In humans, no significant changes in mitochondrial respiration were detected in intact platelets; however, decreased complex I-linked respiration was observed after statin treatment in permeabilized platelets. We propose that the small in vivo effect of statins on platelet energy metabolism can be attributed to drug effects on complex I of the electron transport system. Both intact and permeabilized platelets can be used as a readily available biological model to study changes in cellular energy metabolism in patients treated with statins.

  2. The effect of oleuropein from olive leaf (Olea europaea) extract on Ca²⁺ homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in HepG2 human hepatoma cells.

    PubMed

    Cheng, Jin-Shiung; Chou, Chiang-Ting; Liu, Yuan-Yuarn; Sun, Wei-Chih; Shieh, Pochuen; Kuo, Daih-Huang; Kuo, Chun-Chi; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-05-01

    Oleuropein, a phenolic compound found in the olive leaf (Olea europaea), has been shown to have biological activities in different models. However, the effects of oleuropein on Ca(2+) homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in liver cells have not been analyzed. Oleuropein induced [Ca(2+)]i rises only in HepG2 cells but not in AML12, HA22T or HA59T cells due to the different status of 3-hydroxy-3-methylglutaryl-CoA reductase expression. In HepG2 cells, this Ca(2+) signaling response was reduced by removing extracellular Ca(2+), and was inhibited by the store-operated Ca(2+) channel blockers 2-APB and SKF96365. In Ca(2+)-free medium, pretreatment with the ER Ca(2+) pump inhibitor thapsigargin abolished oleuropein-induced [Ca(2+)]i rises. Oleuropein induced cell cycle arrest which was associated with the regulation of p53, p21, CDK1 and cyclin B1 levels. Furthermore, oleuropein elevated intracellular ROS levels but reduced GSH levels. Treatment with the intracellular Ca(2+) chelator BAPTA-AM or the antioxidant NAC partially reversed oleuropein-induced cytotoxicity. Together, in HepG2 cells, oleuropein induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through store-operated Ca(2+) channels. Moreover, oleuropein induced Ca(2+)-associated cytotoxicity that involved ROS signaling and cell cycle arrest. This compound may offer a potential therapy for treatment of human hepatoma.

  3. Optimizing cardiovascular outcomes in diabetes mellitus.

    PubMed

    Sobel, Burton E

    2007-09-01

    This article presents a series of take-home statements, compiled by a multidisciplinary steering committee, concerning significant aspects of macrovascular disease in patients with diabetes mellitus, including the extent of risk, pathogenetic mechanisms, and optimal management for risk reduction. The discussion focuses in particular on the impact of diabetes medications beyond blood glucose control. In summary, these statements are as follows: (1) Patients with diabetes have an increased risk for cardiovascular disease that contributes to decreased life expectancy; (2) prognosis after a cardiovascular event is poorer in patients with diabetes; (3) pathogenetic mechanisms include insulin resistance, endothelial dysfunction, dyslipidemia, chronic inflammation, procoagulability, and impaired fibrinolysis; (4) management of established cardiovascular risk factors, for example with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) and antihypertensive therapy, reduces cardiovascular event rates in diabetes; (5) correction of hyperglycemia can reduce macrovascular event rates, but the coupling to hyperglycemia is less tight for macrovascular events than it is for reduction of microvascular complications; (6) patients with diabetes should be screened for additional cardiovascular risk factors and appropriate interventions should be initiated; (7) results of observational and interventional studies have indicated that some insulin sensitizers appear to reduce the incidence of cardiovascular events and improve survival; (8) thiazolidinediones have beneficial effects on metabolism that may improve cardiovascular risk, and a randomized clinical trial in patients with advanced atherosclerosis indicates that addition of pioglitazone to therapy for hyperglycemia may reduce the incidence of cardiovascular events such as myocardial infarction and stroke.

  4. A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

    PubMed Central

    Bhattacharya, Bonhi S.; Sweby, Peter K.; Minihane, Anne-Marie; Jackson, Kim G.; Tindall, Marcus J.

    2014-01-01

    Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in a hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main regulator of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homeostasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature. PMID:24444765

  5. Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line.

    PubMed

    Marcuzzi, Annalisa; Zanin, Valentina; Piscianz, Elisa; Tricarico, Paola Maura; Vuch, Josef; Girardelli, Martina; Monasta, Lorenzo; Bianco, Anna Monica; Crovella, Sergio

    2012-10-01

    Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD. We demonstrate that apoptosis in neuronal lovastatin treated-cells is induced by the mitochondrial pathway, with caspase-9 as the initiator and caspase-3 as the effector caspase. The presence of geranylgeraniol modulates both the caspase-9 and caspase-3 activity in a dose-dependent way, confirming that this isoprenoid enters the mevalonate pathway, is metabolized and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. According to our findings, it should not be the time course adopted that modulates the apoptotic response but rather the isoprenoid itself. Being aware that our results have been obtained using a biochemical model of MKD, and not cells from patients with the disease, we believe our findings increase the knowledge of MA pathogenesis, and may possibly contribute to the development of novel therapeutic strategies.

  6. Cross sections for production of the CO(A 1 Pi)-(X 1 Sigma) fourth positive band system and O(3 S) by photodissociation of CO2

    NASA Technical Reports Server (NTRS)

    Gentieu, E. P.; Mentall, J. E.

    1972-01-01

    The CO(A 1 Pi) cross sections reported here, along with previously determined electron impact results, establish the basis for calculating CO fourth positive system volume emission rates in the Martian dayglow. Calculated volume emission rates in turn determine relative distribution of photon vs. electron impact as mechanisms for producing CO(A 1 Pi) in the Mars atmosphere. The smallness of the O(1304) cross section confirms previous indirect evidence that photodissociative excitation of CO2 is not an important source of O(3 S) in the upper atmosphere of Mars.

  7. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  8. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J.; Lewis, Stephen J.

    2015-01-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions. PMID:26637637

  9. Characterization of the JWST Pathfinder mirror dynamics using the center of curvature optical assembly (CoCOA)

    NASA Astrophysics Data System (ADS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-07-01

    The James Webb Space Telescope (JWST) Optical Telescope Element (OTE) consists of a 6.6 m clear aperture, 18 segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at the National Aeronautics and Space Administration (NASA) Johnson Space Center (JSC) using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  10. Characterization of the JWST Pathfinder Mirror Dynamics Using the Center of Curvature Optical Assembly (CoCOA)

    NASA Technical Reports Server (NTRS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-01-01

    The JWST (James Webb Space Telescope) Optical Telescope Element (OTE) consists of a 6.6 meter clear aperture, 18-segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at NASA Johnson Space Center using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  11. Linear magnetoelectric effect as a signature of long-range collinear antiferromagnetic ordering in the frustrated spinel CoA l2O4

    NASA Astrophysics Data System (ADS)

    Ghara, Somnath; Ter-Oganessian, N. V.; Sundaresan, A.

    2017-03-01

    The ground state of the frustrated A -site magnetic spinel CoA l2O4 has been a controversial issue whether it is a collinear antiferromagnetic ordering or a spiral spin-liquid state, as the ratio of the two competing interactions J2/J1 lies close to the boundary between these two ground states. Here we address the magnetic ground state in CoA l2O4 with different amounts of C o2 +/A l3 + site disorder from the study of magnetoelectric effect and Monte Carlo simulations. CoA l2O4 with low site disorder exhibits a linear magnetoelectric effect below the magnetic ordering temperature. With increasing disorder, the magnetoelectric effect is suppressed and the sample with 14 % disorder exhibits a spin glass behavior without the magnetoelectric effect. Monte Carlo simulations support the experimental findings and suggest that the site disorder suppresses long-range antiferromagnetic order and induces a spin glass state. Since the linear magnetoelectric effect requires a long-range magnetic ordering, we suggest that the ground state of CoA l2O4 with low site disorder is a collinear antiferromagnet.

  12. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

  13. FRUCTOSE-6-PHOSPHATE REDUCTASE FROM SALMONELLA GALLINARUM

    PubMed Central

    Zancan, Glaci T.; Bacila, Metry

    1964-01-01

    Zancan, Glaci T. (Universidade do Paraná, Curitiba, Paraná, Brazil), and Metry Bacila. Fructose-6-phosphate reductase from Salmonella gallinarum. J. Bacteriol. 87:614–618. 1964.—A fructose-6-phosphate reductase present in cell-free extracts of Salmonella gallinarum was purified approximately 42 times. The optimal pH for this enzyme is 8.0. The enzyme is specific for fructose-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). The dissociation constants are 1.78 × 10−4m for fructose-6-phosphate and 8.3 × 10−5m for NADH. The Q10, reaction order, and equilibrium constant were determined. The enzyme is sensitive to p-chloromercuribenzoic acid, but not to o-iodosobenzoic acid nor to N-ethylmaleimide. PMID:14127579

  14. Characterization of erythrose reductases from filamentous fungi

    PubMed Central

    2013-01-01

    Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected. PMID:23924507

  15. Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells.

    PubMed

    ElAzzouny, Mahmoud A; Evans, Charles R; Burant, Charles F; Kennedy, Robert T

    2015-01-01

    AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.

  16. Reduced fat mass in rats fed a high oleic acid-rich safflower oil diet is associated with changes in expression of hepatic PPARalpha and adipose SREBP-1c-regulated genes.

    PubMed

    Hsu, Shan-Ching; Huang, Ching-Jang

    2006-07-01

    PPARs and sterol regulatory element-binding protein-1c (SREPB-1c) are fatty acid-regulated transcription factors that control lipid metabolism at the level of gene expression. This study compared a high oleic acid-rich safflower oil (ORSO) diet and a high-butter diet for their effect on adipose mass and expressions of genes regulated by PPAR and SREPB-1c in rats. Four groups of Wistar rats were fed 30S (30% ORSO), 5S (5% ORSO), 30B (29% butter + 1% ORSO), or 5B (4% butter plus 1% ORSO) diets for 15 wk. Compared with the 30B group, the 30S group had less retroperitoneal white adipose tissue (RWAT) mass and lower mRNA expressions of lipoprotein lipase, adipocyte fatty acid-binding protein, fatty acid synthase, acetyl CoA carboxylase, and SREBP-1c in the RWAT, higher mRNA expressions of acyl CoA oxidase, carnitine palmitoyl-transferase 1A, fatty acid binding protein, and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in the liver (P < 0.05). The 18:2(n-6) and 20:4(n-6) contents in the liver and RWAT of the 30S group were >2 fold those of the 30B group (P < 0.05). These results suggested that the smaller RWAT mass in rats fed the high-ORSO diet might be related to the higher tissue 18:2(n-6) and 20:4(n-6). This in turn could upregulate the expressions of fatty acid catabolic genes through the activation of PPARalpha in the liver and downregulate the expressions of lipid storage and lipogenic gene through the suppression of SREBP-1c in the RWAT.

  17. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism. PMID:26536144

  18. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  19. Structure of Coenzyme A-Disulfide Reductase from Staphylococcus aureus at 1.54 Angstrom Resolution

    SciTech Connect

    Mallett,T.; Wallen, J.; Karplus, P.; Sakai, H.; Tsukihara, T.; Claiborne, A.

    2006-01-01

    Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 {angstrom}. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S{gamma} located at the flavin si face, 3.2 {angstrom} from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 {angstrom} from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

  20. Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis

    PubMed Central

    Bomati, Erin K.; Austin, Michael B.; Bowman, Marianne E.; Dixon, Richard A.; Noel, Joseph P.

    2010-01-01

    4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/ketoreductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase. PMID:15970585

  1. Limited proteolysis of the nitrate reductase from spinach leaves.

    PubMed

    Kubo, Y; Ogura, N; Nakagawa, H

    1988-12-25

    The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.

  2. Defective Pollen Wall is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

    SciTech Connect

    Shi, J.; Shanklin, J.; Tan, H.; Yu, X.-H.; Liu, Y.; Liang, W.; Ranathunge, K.; Franke, R. B.; Schreiber, L.; Wang, Y.; Kai, G.; Ma, H.; Zhang, D.

    2011-06-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

  3. Defective pollen wall is required for anther and microspore development in rice and encodes a fatty acyl carrier protein reductase.

    PubMed

    Shi, Jing; Tan, Hexin; Yu, Xiao-Hong; Liu, Yuanyun; Liang, Wanqi; Ranathunge, Kosala; Franke, Rochus Benni; Schreiber, Lukas; Wang, Yujiong; Kai, Guoying; Shanklin, John; Ma, Hong; Zhang, Dabing

    2011-06-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

  4. Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa

    PubMed Central

    Guo, Dianjing; Chen, Fang; Inoue, Kentaro; Blount, Jack W.; Dixon, Richard A.

    2001-01-01

    Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in β-5 linked dimers of G units. Accumulation of soluble caffeic acid β-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis. PMID:11158530

  5. Enzyme toolbox: novel enantiocomplementary imine reductases.

    PubMed

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities.

  6. Soluble ascorbate free radical reductase in the human lens.

    PubMed

    Bando, M; Obazawa, H

    1994-01-01

    A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.

  7. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  8. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development.

    PubMed

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J

    2012-06-01

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT-encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T-DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol-localized, mevalonate-derived isoprenoid biosynthetic pathway.

  9. High doses of atorvastatin and simvastatin induce key enzymes involved in VLDL production.

    PubMed

    Roglans, Núria; Verd, Joan C; Peris, Cristina; Alegret, Marta; Vázquez, Manuel; Adzet, Tomás; Díaz, Cristina; Hernández, Gonzalo; Laguna, Juan C; Sánchez, Rosa M

    2002-05-01

    Treatments with high doses of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors may induce the expression of sterol regulatory element binding protein (SREBP)-target genes, causing different effects from those attributed to the reduction of hepatic cholesterol content. The aim of this study was to investigate the effects of high doses of statins on the key enzymes involved in VLDL production in normolipidemic rats. To examine whether the effects caused by statin treatment are a consequence of HMG-CoA reductase inhibition, we tested the effect of atorvastatin on these enzymes in mevalonate-fed rats. Atorvastatin and simvastatin enhanced not only HMG-CoA reductase but also the expression of the SREBP-2 gene itself. As a result of the overexpression of SREBP-2 caused by the statin treatment, genes regulated basically by SREBP-1, as FA synthase and acetyl-coenzyme A carboxylase, were also induced and their mRNA levels increased. DAG acyltransferase and microsomal TG transfer protein mRNA levels as well as phosphatidate phosphohydrolase activity were increased by both statins. Simvastatin raised liver cholesterol content, ACAT mRNA levels, and CTP:phosphocholine cytidylyltransferase activity, whereas it reduced liver DAG and phospholipid content. Mevalonate feeding reversed all changes induced by the atorvastatin treatment. These results show that treatment with high doses of statins induces key enzymes controlling rat liver lipid synthesis and VLDL assembly, probably as a result of SREBP-2 overexpression. Despite the induction of the key enzymes involved in VLDL production, both statins markedly reduced plasma TG levels, suggesting that different mechanisms may be involved in the hypotriglyceridemic effect of statins at high or low doses.

  10. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  11. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  12. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    PubMed Central

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  13. Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

    PubMed

    Ikegawa, S; Goto, T; Mano, N; Goto, J

    1998-11-01

    The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

  14. Methylenetetrahydrofolate reductase: biochemical characterization and medical significance.

    PubMed

    Trimmer, Elizabeth E

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). The enzyme employs a noncovalently-bound flavin adenine dinucleotide (FAD), which accepts reducing equivalents from NAD(P)H and transfers them to CH2-H4folate. The reaction provides the sole source of CH3-H4folate, which is utilized by methionine synthase in the synthesis of methionine from homocysteine. MTHFR plays a key role in folate metabolism and in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia. A common C677T polymorphism in MTHFR has been associated with an increased risk for the development of cardiovascular disease, Alzheimer's disease, and depression in adults, and of neural tube defects in the fetus. The mutation also confers protection for certain types of cancers. This review presents the current knowledge of the enzyme, its biochemical characterization, and medical significance.

  15. Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

    PubMed Central

    Ohura, T; Kraus, J P; Rosenberg, L E

    1989-01-01

    We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2741949

  16. Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity.

    PubMed

    Vaidyanathan, Ramanathan; Gopalram, Shubaash; Kalishwaralal, Kalimuthu; Deepak, Venkataraman; Pandian, Sureshbabu Ram Kumar; Gurunathan, Sangiliyandi

    2010-01-01

    Nanostructure materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. The generation of silver nanoparticles using optimized nitrate reductase for the reduction of Ag(+) with the retention of enzymatic activity in the complex is being reported. This report involves the optimization of enzyme activity to bring about enhanced nanoparticle synthesis. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of nitrate reductase by Bacillus licheniformis at pH 8. The four variables involved in the study of nitrate reductase were Glucose, Peptone, Yeast extract and KNO(3). Glucose had a significant effect on nitrate reductase production. The optimized medium containing (%) Glucose: 1.5, Peptone: 1, Yeast extract: 0.35 and KNO(3): 0.35 resulted in a nitrate reductase activity of 452.206 U/ml which is same as that of the central level. The medium A (showing least nitrate reductase activity) and the medium B (showing maximum nitrate reductase activity) were compared for the synthesis. Spectrophotometric analysis revealed that the particles exhibited a peak at 431 nm and the A(431) for the medium B was 2-fold greater than that of the medium A. The particles were also characterized using TEM. The particles synthesized using the optimized enzyme activity ranged from 10 to 80 nm and therefore can be extended to various medicinal applications.

  17. Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

    PubMed Central

    Igarashi, Junsuke; Hashimoto, Takeshi; Kubota, Yasuo; Shoji, Kazuyo; Maruyama, Tokumi; Sakakibara, Norikazu; Takuwa, Yoh; Ujihara, Yoshihiro; Katanosaka, Yuki; Mohri, Satoshi; Naruse, Keiji; Yamashita, Tetsuo; Okamoto, Ryuji; Hirano, Katsuya; Kosaka, Hiroaki; Takata, Maki; Konishi, Ryoji; Tsukamoto, Ikuko

    2014-01-01

    COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca2+ concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [3H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis. PMID:25505610

  18. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    PubMed

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  19. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  20. COA User's Guide

    SciTech Connect

    Fox, B.; Pautz, J.; Sellers, C.

    1999-01-28

    The Department of Energy (DOE) has one of the largest and most complete collections of information on crude oil composition that is available to the public. The computer program that manages this database of crude oil analyses has recently been rewritten to allow easier access to this information. This report describes how the new system can be accessed and how the information contained in the Crude Oil Analysis Data Bank can be obtained.

  1. Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

    PubMed

    Gamenara, Daniela; Domínguez de María, Pablo

    2014-05-21

    Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

  2. Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis

    PubMed Central

    Abreu, Vinícius; Diniz, Carlos; Dorneles, Elaine M. S.; Barh, Debmalya

    2017-01-01

    Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets. PMID:28316974

  3. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... activity of the enzyme glutathione reductase in serum, plasma, or erythrocytes by such techniques as fluorescence and photometry. The results of this assay are used in the diagnosis of liver disease,...

  4. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed

    Sengupta, S; Shaila, M S; Rao, G R

    1996-07-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

  5. Comparative anatomy of the aldo-keto reductase superfamily.

    PubMed

    Jez, J M; Bennett, M J; Schlegel, B P; Lewis, M; Penning, T M

    1997-09-15

    The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short

  6. RNAi inhibition of feruloyl CoA 6'-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots.

    PubMed

    Liu, Shi; Zainuddin, Ima M; Vanderschuren, Herve; Doughty, James; Beeching, John R

    2017-03-18

    Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24-72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava's marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6'-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6'-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6'-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.

  7. Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

    PubMed Central

    Iuchi, S; Lin, E C

    1987-01-01

    In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector. PMID:3301812

  8. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    PubMed

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  9. Carbon-carbon double-bond reductases in nature.

    PubMed

    Huang, Minmin; Hu, Haihong; Ma, Li; Zhou, Quan; Yu, Lushan; Zeng, Su

    2014-08-01

    Reduction of C = C bonds by reductases, found in a variety of microorganisms (e.g. yeasts, bacteria, and lower fungi), animals, and plants has applications in the production of metabolites that include pharmacologically active drugs and other chemicals. Therefore, the reductase enzymes that mediate this transformation have become important therapeutic targets and biotechnological tools. These reductases are broad-spectrum, in that, they can act on isolation/conjugation C = C-bond compounds, α,β-unsaturated carbonyl compounds, carboxylic acids, acid derivatives, and nitro compounds. In addition, several mutations in the reductase gene have been identified, some associated with diseases. Several of these reductases have been cloned and/or purified, and studies to further characterize them and determine their structure in order to identify potential industrial biocatalysts are still in progress. In this study, crucial reductases for bioreduction of C = C bonds have been reviewed with emphasis on their principal substrates and effective inhibitors, their distribution, genetic polymorphisms, and implications in human disease and treatment.

  10. Effect of vitamin D on bioavailability and lipid lowering efficacy of simvastatin.

    PubMed

    Al-Asmari, Abdulrahman K; Ullah, Zabih; Al-Sabaan, Fahad; Tariq, Mohammad; Al-Eid, Ahmed; Al-Omani, Saud F

    2015-03-01

    The 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors known as "statins" are widely prescribed for the management of dyslipidemia. In spite of their muscle toxicity, use of statins has alarmingly increased worldwide. A recent report suggests that vitamin D (VD) levels are closely associated with lipid lowering activity and muscular toxicity of statins. However, data are limited and inconclusive. The present study was undertaken to investigate the effect of VD supplementation on the bioavailability and lipid lowering effect of simvastatin (ST). Adult Sprague-Dawley male rats (250 ± 10 g) were divided into four groups including control, ST (100 mg/kg/day), VD (100 μg/kg/day) and ST + VD group, respectively. After the dosing period of 8 days the animals were sacrificed and the blood was collected for the analysis of ST, its active metabolite simvastatin acid (STA), total cholesterol, triglyceride and liver enzymes including aspartate transaminase and alanine transaminase. The result of this study showed a significant decrease in the level of cholesterol and triglyceride in ST alone treated group, whereas VD alone failed to alter the blood lipid levels. Concomitant treatment with VD produced significant decrease in the bioavailability of ST and STA. However, there was no significant difference in the level of cholesterol in ST alone and in ST + VD treated group. Our results on the liver enzyme suggest that ST alone or in combination with VD does not produce any hepatotoxicity. Further studies using VD along with various statins for a longer duration are suggested.

  11. Results of the primary outcome measure and clinical events from the Asymptomatic Carotid Artery Progression Study.

    PubMed

    Probstfield, J L; Margitic, S E; Byington, R P; Espeland, M A; Furberg, C D

    1995-09-28

    The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have proven to be more effective in reducing levels of low density lipoprotein (LDL) cholesterol and to be better tolerated than other lipid-lowering compounds. Most of the trials evaluating the effects of these new agents on progression of atherosclerosis have not included individuals asymptomatic for cardiovascular disease and who have LDL cholesterol levels at or below the limits established by the National Cholesterol Education Program for initiating treatment. The Asymptomatic Carotid Artery Progression Study (ACAPS) tested the effect of the HMG-CoA reductase inhibitor, lovastatin, on early-stage carotid atherosclerosis (as detected by B-mode ultrasonography) in 919 asymptomatic men and women, 40-79 years of age, who had LDL cholesterol levels between the 60th and 90th percentiles. Participants randomized into this double-blind, placebo-controlled, factorially designed study received lovastatin (20-40 mg/day) or lovastatin-placebo and warfarin (1 mg/day), or warfarin-placebo over a 3-year period. The progression of the mean maximum intimal-medial thickness (IMT) over 12 walls of both carotid arteries represented the primary outcome. Lovastatin treatment was associated with a reduction in progression of mean maximum IMT (p < 0.001). Levels of LDL cholesterol were reduced by 28% (43.5 mg/dl [11.25 mmol/liter]) in the lovastatin group within 6 months (p < 0.0001) and remained stable throughout the follow-up period, whereas these levels remained essentially unchanged in the lovastatin-placebo group. The difference in incidence of major cardiovascular events for patients in the lovastatin-placebo group was significant: 5 versus 14, respectively (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Cinnamon extract improves the body composition and attenuates lipogenic processes in the liver and adipose tissue of rats.

    PubMed

    Lopes, Bruna P; Gaique, Thaiane G; Souza, Luana L; Paula, Gabriela S M; Kluck, George E G; Atella, Georgia C; Gomes, Anne Caroline C; Simas, Naomi K; Kuster, Ricardo M; Ortiga-Carvalho, Tania M; Pazos-Moura, Carmen C; Oliveira, Karen J

    2015-10-01

    In models of metabolic disorders, cinnamon improves glucose and lipid metabolism. This study explores the effect of chronic supplementation with aqueous cinnamon extract (CE) on the lipid metabolism of rats. Male adult Wistar rats were separated into a control group (CTR) receiving water and a CE Group receiving aqueous cinnamon extract (400 mg of cinnamon per kg body mass per day) by gavage for 25 consecutive days. Cinnamon supplementation did not change the food intake or the serum lipid profile but promoted the following changes: lower body mass gain (P = 0.008), lower relative mass of white adipose tissue (WAT) compartments (P = 0.045) and higher protein content (percentage of the carcass) (P = 0.049). The CE group showed lower leptin mRNA expression in the WAT (P = 0.0017) and an important tendency for reduced serum leptin levels (P = 0.059). Cinnamon supplementation induced lower mRNA expression of SREBP1c (sterol regulatory element-binding protein 1c) in the WAT (P = 0.001) and liver (P = 0.013) and lower mRNA expression of SREBP2 (P = 0.002), HMGCoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (P = 0.0003), ACAT1 (acetyl-CoA acetyltransferase 1) (P = 0.032) and DGAT2 (diacylglycerol O-acyltransferase 2) (P = 0.03) in the liver. These changes could be associated with the reduced esterified cholesterol and triacylglycerol content detected in this tissue. Our results suggest that chronic ingestion of aqueous cinnamon extract attenuates lipogenic processes, regulating the expression of key enzymes and transcriptional factors and their target genes, which are directly involved in lipogenesis. These molecular changes possibly promote adaptations that would prevent an increase in circulating cholesterol and triacylglycerol levels and prevent lipid accumulation in tissues, such as liver and WAT. Therefore, we speculate that cinnamon may also be useful for preventing or retarding the development of lipid disorders.

  13. Management of dyslipidemia and hyperglycemia with a fixed-dose combination of sitagliptin and simvastatin

    PubMed Central

    Steinberg, Helmut; Anderson, Matt S; Musliner, Thomas; Hanson, Mary E; Engel, Samuel S

    2013-01-01

    The risk of death due to heart disease and stroke is up to four times higher in individuals with diabetes compared to individuals without diabetes. Most guidelines that address treatment of dyslipidemia in patients with diabetes consider diabetes a cardiovascular disease (CVD) “risk equivalent” and recommend intensive treatment of dyslipidemia for the purpose of CVD prevention. Statins (3-hydroxy 3-methylglutaryl coenzyme A reductase [HMG-CoA reductase] inhibitors) are first-line agents in achieving lipid goals as an adjunct to diet and exercise and should be used in most patients. In addition to lipid management and blood pressure control, glycemic control is a basic component in the management of diabetes. Glycemic control is achieved by combining diabetes self-management education, diet and exercise, and, where required, antihyperglycemic agents (OHAs). Persistence and adherence to therapy are critical in achieving recommended treatment goals. However, overall compliance with concomitantly prescribed OHAs and statins is low in patients with type 2 diabetes. Fixed-dose combination (FDC) therapies have been shown to improve adherence by reducing pill burden, the complexity of treatment regimen, and, potentially, cost. Based on the available evidence regarding the pharmacokinetics and the efficacy and safety profiles of each component drug, the sitagliptin/simvastatin FDC may provide a rational and well-tolerated approach to achieving better adherence to multiple-drug therapy and improved lipid lowering and glycemic control, with consequent reduction in cardiovascular risk, diabetic microvascular disease, and mortality in diabetic patients for whom treatment with both compounds is appropriate. PMID:23761972

  14. High-dose statin use does not impair aerobic capacity or skeletal muscle function in older adults

    PubMed Central

    Stock, Anthoney A.; Harman, S. Mitchell

    2008-01-01

    3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are lipid-lowering agents widely employed for atherosclerosis prevention. HMG-CoA reductase blockade reduces skeletal muscle coenzyme Q10 (CoQ10) levels and mitochondrial respiratory chain activities and may produce mild to severe skeletal muscle myopathy. This study investigated whether high-dose statin treatment would result in measurably decreased exercise capacity in older men and women. Maximal oxygen consumption, aerobic endurance, oxygen uptake kinetics, maximal strength, muscular power, and muscular endurance were measured before and after 12 weeks of statin treatment (simvastatin, 80 mg/day) in nine men and one woman, ages 55–76 years, with LDL-cholesterol levels >3.3 mmol/l (mean = 4.2 ± 0.2 mmol/l). Myalgia symptoms were assessed every 4 weeks. As expected, statin treatment resulted in significant decreases in LDL- and total-cholesterol levels (P < 0.01) with no significant changes in HDL-cholesterol or triglyceride levels. No significant changes were observed in aerobic capacity, endurance, oxygen kinetics or any measures of muscle function. No subject reported symptoms of myalgia, cramps, or weakness during the study. In the absence of myalgia or myopathic symptoms, high-dose simvastatin treatment did not impair exercise capacity in hyperlipidemic older individuals. We conclude that decreases in intramuscular CoQ10, in most patients on high dose statin treatment may not be clinically relevant, due to inter-individual variability in the degree of CoQ10 depletion, sensitivity of muscle to decreases in CoQ10, or both. PMID:19424852

  15. Asbestos induces nitric oxide synthesis in mesothelioma cells via Rho signaling inhibition.

    PubMed

    Riganti, Chiara; Orecchia, Sara; Silvagno, Francesca; Pescarmona, Gianpiero; Betta, Pier Giacomo; Gazzano, Elena; Aldieri, Elisabetta; Ghigo, Dario; Bosia, Amalia

    2007-06-01

    We have observed that in three human malignant mesothelioma cell lines, crocidolite asbestos induced the activation of the transcription factor NF-kappaB and the synthesis of nitric oxide (NO) by inhibiting the RhoA signaling pathway. The incubation with crocidolite decreased the level of GTP-bound RhoA and the activity of Rho-dependent kinase, and induced the activation of Akt/PKB and IkBalpha kinase, leading to the nuclear translocation of NF-kappaB. The effects of crocidolite fibers on NF-kappaB activation and NO synthesis were mimicked by Y27632 (an inhibitor of the Rho-dependent kinases) and toxin B (an inhibitor of RhoA GTPase activity), while they were reverted by mevalonic acid, the product of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. Furthermore, crocidolite, similarly to mevastatin, inhibited the synthesis of cholesterol and ubiquinone and the prenylation of RhoA: these effects were prevented in the presence of mevalonic acid. This suggests that crocidolite fibers might inhibit the synthesis of isoprenoid molecules at the level of the HMGCoA reductase reaction or of an upstream step, thus impairing the prenylation and subsequent activation of RhoA. Akt can stimulate NO synthesis via a double mechanism: it can activate the inducible NO synthase via the NF-kappaB pathway and the endothelial NO synthase via a direct phosphorylation. Our results suggest that crocidolite increases the NO levels in mesothelioma cells by modulating both NO synthase isoforms.

  16. High molecular weight poly-gamma-glutamic acid regulates lipid metabolism in rats fed a high-fat diet and humans.

    PubMed

    Park, Ji Ho; Choi, Jae-Chul; Sung, Moon-Hee; Kang, Jae-Heon; Chang, Moon-Jeong

    2011-07-01

    We investigated the effect of high molecular weight polygamma- glutamic acid (hm gamma-PGA) on adiposity and lipid metabolism of rats in the presence of an obesity-inducing diet. Thirty-two Sprague-Dawley rats were fed either a normal-fat (11.4% kcal fat, NFC) or high-fat (51% kcal fat, HFC) diet. After 5 weeks, half of each diet-fed group was treated with hm gamma-PGA (NFP or HFP) for 4 weeks. The HFC group had significantly higher body weight, visceral fat mass, fasting serum levels of total cholesterol, LDL cholesterol, and leptin, and lower serum HDL cholesterol level compared with those of the NFC group (p < 0.05). Treatment with hm gamma-PGA decreased body weight gain and perirenal fat mass (p<0.05), fasting serum total cholesterol, and mRNA expression of glucose-6- phosphate dehydrogenase (G6PD), regardless of dietary fat contents (p < 0.01). However, hm gamma-PGA increased serum HDL cholesterol in the HFC group (p < 0.05). In vitro, 3-hydroxy-3-methylglutaryl coenzyme-A (HMGCoA) reductase activity was suppressed by the addition of hm gamma-PGA. In agreement with observations in animal study, the supplementation of hm gamma-PGA (150 mg/day) to 20 female subjects in an 8-week double-blind, placebocontrolled study resulted in a tendency to decrease total cholesterol and LDL cholesterol concentrations. We thus conclude that dietary supplementation of hm gamma-PGA may act as a hypocholestrolemic agent, secondary to its inhibitor effect on HMG-CoA reductase, and decrease abdominal adiposity by decreasing hepatic lipogenesis. The present study is an important first step in establishing the effect of hm gamma-PGA on cholesterol levels in rats and humans.

  17. Hypocholesterolemic Effects of the Cauliflower Culinary-Medicinal Mushroom, Sparassis crispa (Higher Basidiomycetes), in Diet-Induced Hypercholesterolemic Rats.

    PubMed

    Hong, Ki Bae; Hong, Sung-Yong; Joung, Eun Young; Kim, Byung Hee; Bae, Song-Hwan; Park, Yooheon; Suh, Hyung Joo

    2015-01-01

    The cauliflower culinary-medicinal mushroom, Sparassis crispa, possesses various biological activities that have been widely reported to have therapeutic applications. We examined the effects of S. crispa on serum cholesterol, hepatic enzymes related to cholesterol metabolism, and fecal sterol excretion in rats fed a cholesterol-rich diet for 4 weeks. Male Sprague-Dawley rats (8 weeks old) were randomly divided into 5 groups (n = 6 mice per group): normal diet (normal control [NC]), cholesterol-rich diet (cholesterol control [CC]), cholesterol-rich diet plus S. crispa fruiting body (SC), cholesterol-rich diet plus S. crispa extract (SCE), and cholesterol-rich diet plus S. crispa residue (SCR). SCE supplementation significantly enhanced hepatic cholesterol catabolism through the upregulation of cholesterol 7α-hydroxylase (CYP7A1) messenger RNA (mRNA) expression (2.55-fold compared with that in the NC group; P < 0.05) and the downregulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA expression (0.57-fold compared with that in the NC group; P < 0.05). Additionally, the SCE diet resulted in the highest fecal excretion of cholesterol and bile acid in hypercholesterolemic rats. In conclusion, mRNA expression of CYP7A1 and HMG-CoA reductase were significantly modulated by the absorption of SCE samples. Also, SCE samples had a significant effect on fecal bile acid and cholesterol excretion. These results suggest that SCE samples can induce hypocholesterolic effects through cholesterol metabolism and the reduction of circulating cholesterol levels.

  18. 248-nm laser photolysis of CHBr3/O-atom mixtures: kinetic evidence for UV CO(A) chemiluminescence in the reaction of methylidyne radicals with atomic oxygen.

    PubMed

    Vaghjiani, Ghanshyam L

    2005-03-17

    The 4th positive and Cameron band emissions from electronically excited CO have been observed for the first time in 248-nm pulsed laser photolysis of a trace amount of CHBr(3) vapor in an excess of O atoms. O atoms were produced by dissociation of N(2)O (or O(2)) in a cw-microwave discharge cavity in 2.0 Torr of He at 298 K. The CO emission intensity in these bands showed a quadratic dependence on the laser fluence employed. Temporal profiles of the CO(A) and other excited-state products that formed in the photoproduced precursor + O-atom reactions were measured by recording their time-resolved chemiluminescence in discrete vibronic bands. The CO 4th positive transition (A(1)Pi, v' = 0 --> X(1)Sigma(+), v' ' = 2) near 165.7 nm was monitored in this work to deduce the pseudo-first-order decay kinetics of the CO(A) chemiluminescence in the presence of various added substrates (CH(4), NO, N(2)O, H(2), and O(2)). From this, the second-order rate coefficient values were determined for reactions of these substrates with the photoproduced precursors. The measured reactivity trends suggest that the prominent precursors responsible for the CO(A) chemiluminescence are the methylidyne radicals, CH(X(2)Pi) and CH(a(4)Sigma(-)), whose production requires the absorption of at least 2 laser photons by the photolysis mixture. The O-atom reactions with brominated precursors (CBr, CHBr, and CBr(2)), which also form in the photolysis, are shown to play a minor role in the production of the CO(A or a) chemiluminescence. However, the CBr(2) + O-atom reaction was identified as a significant source for the 289.9-nm Br(2) chemiluminescence that was also observed in this work. The 282.2-nm OH and the 336.2-nm NH chemiluminescences were also monitored to deduce the kinetics of CH(X(2)Pi) and CH(a(4)Sigma(-)) reactions when excess O(2) and NO were present.

  19. Microsecond subdomain folding in dihydrofolate reductase.

    PubMed

    Arai, Munehito; Iwakura, Masahiro; Matthews, C Robert; Bilsel, Osman

    2011-07-08

    The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

  20. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  1. Sulfite reductase protects plants against sulfite toxicity.

    PubMed

    Yarmolinsky, Dmitry; Brychkova, Galina; Fluhr, Robert; Sagi, Moshe

    2013-02-01

    Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum 'Rheinlands Ruhm') and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

  2. Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols.

    PubMed

    Doan, Thuy T P; Carlsson, Anders S; Stymne, Sten; Hofvander, Per

    2016-01-01

    Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

  3. Defective Pollen Wall Is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase[C][W][OA

    PubMed Central

    Shi, Jing; Tan, Hexin; Yu, Xiao-Hong; Liu, Yuanyun; Liang, Wanqi; Ranathunge, Kosala; Franke, Rochus Benni; Schreiber, Lukas; Wang, Yujiong; Kai, Guoying; Shanklin, John; Ma, Hong; Zhang, Dabing

    2011-01-01

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots. PMID:21705642

  4. Uterine glutathione reductase activity: modulation by estrogens and progesterone.

    PubMed

    Díaz-Flores, M; Baiza-Gutman, L A; Pedrón, N N; Hicks, J J

    1999-10-29

    The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.

  5. Bacterial morphinone reductase is related to Old Yellow Enzyme.

    PubMed Central

    French, C E; Bruce, N C

    1995-01-01

    Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine. The structural gene encoding morphinone reductase, designated morB, was cloned from Ps. putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme. Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp. and the related oestrogen-binding protein of Candida albicans. Expressed sequence tags from several plant species