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Sample records for cholesterol oxidase physiological

  1. Physiological and pathological implications of cholesterol.

    PubMed

    Cortes, Victor A; Busso, Dolores; Maiz, Alberto; Arteaga, Antonio; Nervi, Flavio; Rigotti, Attilio

    2014-01-01

    Cholesterol has evolved to fulfill sophisticated biophysical, cell signaling and endocrine requirements of animal systems. At a cellular level, cholesterol is found in membranes, where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains with critical topographical and signaling functions. At an organismal level, cholesterol is the precursor for all steroid hormones, including gluco- and mineralo-corticoids, sex hormones and vitamin D, all of which regulate carbohydrate, sodium, reproductive and bone homeostasis, respectively. This sterol is also the precursor for bile acids, which are important for intestinal absorption of dietary lipids as well as energy and glucose metabolic regulation. Importantly, complex mechanisms maintain cholesterol within physiological ranges and the disregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these diseases has been demonstrated by diverse genetic and pharmacologic animal models that are commented in this review. PMID:24389193

  2. Physiological and pathological implications of cholesterol.

    PubMed

    Cortes, Victor A; Busso, Dolores; Maiz, Alberto; Arteaga, Antonio; Nervi, Flavio; Rigotti, Attilio

    2014-01-01

    Cholesterol has evolved to fulfill sophisticated biophysical, cell signaling and endocrine requirements of animal systems. At a cellular level, cholesterol is found in membranes, where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains with critical topographical and signaling functions. At an organismal level, cholesterol is the precursor for all steroid hormones, including gluco- and mineralo-corticoids, sex hormones and vitamin D, all of which regulate carbohydrate, sodium, reproductive and bone homeostasis, respectively. This sterol is also the precursor for bile acids, which are important for intestinal absorption of dietary lipids as well as energy and glucose metabolic regulation. Importantly, complex mechanisms maintain cholesterol within physiological ranges and the disregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these diseases has been demonstrated by diverse genetic and pharmacologic animal models that are commented in this review.

  3. An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase

    PubMed Central

    Pundir, C.S.; Narang, Jagriti; Chauhan, Nidhi; Sharma, Preety; Sharma, Renu

    2012-01-01

    Background & objectives: The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Methods: Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. Results: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. Km and Imax for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4±2.8 and 92.3±3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were <2 and <4 per cent, respectively. Biosensor had a storage life of 6 months at 4°C. Interpretation & conclusions: The use of epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential. PMID:23168704

  4. Implementation of cellulomonas cholesterol oxidase for total serum cholesterol determination by the endpoint method.

    PubMed

    Srisawasdi, Pornpen; Chaichanajarernkul, Upsorn; Teerakranjana, Narumon; Kroll, Martin H

    2008-01-01

    Cellulomonas has been shown to be a good source of cholesterol oxidase in addition to Streptomyces for serum cholesterol determination by the endpoint method, inexpensive in cost, and showing excellent performance. For clinical use, we have assessed the reliability of Cellulomonas reagent for cholesterol determination. We constructed the user-defined endpoint methods on three automated analyzers. The analytical performances (linearity, precision, recovery, interference, stability, and comparison with the standardized method) of Cellulomonas cholesterol reagents were evaluated and compared to those of Streptomyces reagents. Linearity (18.1-23.3 mmol/L) and stability of reagents (6-11 weeks) depended on the analyzers being used. The average within-run and between-day % coefficients of variation (CVs) ranged from 1.44 to 2.45 and 1.98 to 2.99, respectively, and were within National Cholesterol Education Program analytical criteria (cholesterol recovery while lipemia generated a positive interference with all methods. Cellulomonas enzyme is analytically reliable when used for serum cholesterol determination by the endpoint method. Its analytical performance is equivalent to Streptomyces enzymes and meets the analytical goals. It has an advantage over the other enzymes in that it does not ship in the frozen state.

  5. Cholesterol oxidase catalyzed oxidation of cholesterol in mixed lipid monolayers: effects of surface pressure and phospholipid composition on catalytic activity.

    PubMed

    Grönberg, L; Slotte, J P

    1990-04-01

    The catalytic activity of cholesterol oxidase from Streptomyces sp. in mixed monolayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), N-oleoylsphingomyelin (O-SPM), and cholesterol (CHL) has been determined at lateral surface pressures between 10 and 30 mN/m. The highest cholesterol oxidase activity (determined at 37 degrees C) was observed at surface pressures around 20 mN/m in a POPC/CHL monolayer (50:50 mol %). Above and below this surface pressure, the enzyme activity decreased markedly. A similar optimal activity vs surface pressure relationship was observed also for an O-SPM/CHL monolayer (50:50 mol %). The activity of cholesterol oxidase toward cholesterol in the O-SPM/CHL monolayer was, however, less than in the corresponding POPC mixed monolayer. The surface activity of cholesterol oxidase decreased markedly when the temperature was lowered to 20 degrees C, and hardly any enzyme activity was observed in an O-SPM/CHL monolayer at 25 mN/m or above. With a monolayer containing POPC/O-SPM/CHL (42:18:40 mol %), maximal cholesterol oxidase activity was observed at the lowest surface pressure tested (i.e., 10 mN/m), and the catalytic activity decreased markedly with increasing lateral surface pressures in the monolayer. The results of this study show (i) that the activity of cholesterol oxidase in general is highly dependent on the lateral surface pressure in the substrate membranes and (ii) that sphingomyelin, by interacting tightly with cholesterol, can prevent or restrain the accessibility of cholesterol for oxidation by cholesterol oxidase.

  6. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    PubMed Central

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  7. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  8. Active membrane cholesterol as a physiological effector.

    PubMed

    Lange, Yvonne; Steck, Theodore L

    2016-09-01

    Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily.

  9. Physiological and pathological implications of semicarbazide-sensitive amine oxidase.

    PubMed

    Yu, Peter H; Wright, Shannon; Fan, Ellen H; Lun, Zhao-Rong; Gubisne-Harberle, Diana

    2003-04-11

    Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of primary amines. Such deamination has been shown capable of regulating glucose transport in adipose cells. It has been independently discovered that the primary structure of vascular adhesion protein-1 (VAP-1) is identical to SSAO. VAP-1 regulates leukocyte migration and is related to inflammation. Increased serum SSAO activities have been found in patients with diabetic mellitus, vascular disorders and Alzheimer's disease. The SSAO-catalyzed deamination of endogenous substrates, that is, methylamine and aminoacetone, led to production of toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia, respectively. These highly reactive aldehydes have been shown to initiate protein cross-linkage, exacerbate advanced glycation of proteins and cause endothelial injury. Hydrogen peroxide contributes to oxidative stress. 14C-methylamine is converted to 14C-formaldehyde, which then forms labeled long-lasting protein adduct in rodents. Chronic methylamine treatment increased the excretion of malondialdehyde and microalbuminuria, and enhanced the formation of fatty streaks in C57BL/6 mice fed with an atherogenic diet. Treatment with selective SSAO inhibitor reduces atherogenesis in KKAy diabetic mice fed with high-cholesterol diet. Aminoguanidine, which blocks advanced glycation and reduces nephropathy in animals, is in fact more potent at inhibiting SSAO than its effect on glycation. It suggests that SSAO is involved in vascular disorders under certain pathological conditions. Although SSAO has been known for several decades, its physiological and pathological implications are just beginning to be recognized.

  10. Retracted: Advances in the physiological and pathological implications of cholesterol.

    PubMed

    Cortes, Victor A; Busso, Dolores; Mardones, Pablo; Maiz, Alberto; Arteaga, Antonio; Nervi, Flavio; Rigotti, Attilio

    2013-11-01

    Cholesterol has evolved to fulfill sophisticated biophysical, cell signalling, and endocrine functions in animal systems. At the cellular level, cholesterol is found in membranes where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains with critical topographical and signalling functions. At the organismal level, cholesterol is the precursor of all steroid hormones, including gluco- and mineralo-corticoids, sex hormones, and vitamin D, which regulate carbohydrate, sodium, reproductive, and bone homeostasis, respectively. This sterol is also the immediate precursor of bile acids, which are important for intestinal absorption of dietary lipids as well as energy homeostasis and glucose regulation. Complex mechanisms maintain cholesterol within physiological ranges and the dysregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these conditions has been demonstrated by genetic and pharmacological manipulations in animal models of human disease that are discussed herein. Importantly, the understanding of basic aspects of cholesterol biology has led to the development of high-impact pharmaceutical therapies during the past century. The continuing effort to offer successful treatments for prevalent cholesterol-related diseases, such as atherosclerosis and neurodegenerative disorders, warrants further interdisciplinary research in the coming decades. PMID:23445165

  11. Retracted: Advances in the physiological and pathological implications of cholesterol.

    PubMed

    Cortes, Victor A; Busso, Dolores; Mardones, Pablo; Maiz, Alberto; Arteaga, Antonio; Nervi, Flavio; Rigotti, Attilio

    2013-11-01

    Cholesterol has evolved to fulfill sophisticated biophysical, cell signalling, and endocrine functions in animal systems. At the cellular level, cholesterol is found in membranes where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains with critical topographical and signalling functions. At the organismal level, cholesterol is the precursor of all steroid hormones, including gluco- and mineralo-corticoids, sex hormones, and vitamin D, which regulate carbohydrate, sodium, reproductive, and bone homeostasis, respectively. This sterol is also the immediate precursor of bile acids, which are important for intestinal absorption of dietary lipids as well as energy homeostasis and glucose regulation. Complex mechanisms maintain cholesterol within physiological ranges and the dysregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these conditions has been demonstrated by genetic and pharmacological manipulations in animal models of human disease that are discussed herein. Importantly, the understanding of basic aspects of cholesterol biology has led to the development of high-impact pharmaceutical therapies during the past century. The continuing effort to offer successful treatments for prevalent cholesterol-related diseases, such as atherosclerosis and neurodegenerative disorders, warrants further interdisciplinary research in the coming decades.

  12. ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014.

    PubMed

    Fernández de Las Heras, Laura; Mascaraque, Victoria; García Fernández, Esther; Navarro-Llorens, Juana María; Perera, Julián; Drzyzga, Oliver

    2011-07-20

    Cholesterol catabolism has been reported in different bacteria and particularly in several Rhodococcus species, but the genetic of this complex pathway is not yet very well defined. In this work we report the isolation and sequencing of a 9.8 kb DNA fragment of Rhodococcus sp. strain CECT3014, a bacterial strain that we here identify as a Rhodococcus erythropolis strain. In this DNA fragment we found several ORF that are probably involved in steroid catabolism, and choG, a gene encoding a putative cholesterol oxidase whose functional characterization we here report. ChoG protein is a class II cholesterol oxidase with all the structural features of the enzymes of this group. The disruption of the choG gene does not alter the ability of strain CECT3014 cells to grow on cholesterol, but it abolishes the production of extracellular cholesterol oxidase. This later effect is reverted when the mutant cells are transformed with a plasmid expressing choG. We conclude that choG is the gene responsible for the inducible extracellular cholesterol oxidase activity of strain CECT3014. This activity distributes between the cellular membrane and the culture supernatant in a way that suggests it is produced by the same ChoG protein that occurs in two different locations. RT-PCR transcript analysis showed a dual scheme of choG expression: a low constitutive independent transcription, plus a cholesterol induced transcription of choG into a polycistronic kstD-hsd4B-choG mRNA. PMID:20630728

  13. Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

    PubMed Central

    Nien, Po-Chin; Chen, Po-Yen; Ho, Kuo-Chuan

    2009-01-01

    In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days. PMID:22573987

  14. Distortion of Flavin Geometry Is Linked to Ligand Binding in Cholesterol Oxidase

    SciTech Connect

    Lyubimov, A.Y.; Heard, K.; Tang, H.; Sampson, N.S.; Vrielink, A.

    2009-06-03

    Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

  15. Cholesterol Oxidase Functionalised Polyaniline/Carbon Nanotube Hybrids for an Amperometric Biosensor.

    PubMed

    Shukla, Sudheesh K; Turner, Anthony P F; Tiwari, Ashutosh

    2015-05-01

    Functional carbon nanotubes (CNT) have attracted much attention for analytical and biomedical applications. This paper describes the fabrication of a cholesterol oxidase (ChOx) immobilised polyaniline (PANI)/CNT composite electrode for the amperometric detection of cholesterol. The prepared ChOx/PANI/CNT/Au bioelectrode bound ChOx via the available functionalties of PANI (-NH2) and CNT (-COOH). Moreover, the CNT creates a network inside the matrix that strengthens the mechanical property of the bioelectrode. The multifunctional matrix is presumed to provide a 3D-mesoporous surface, which substantially enhances enzyme activity. The linear range of the biosensor for cholesterol oleate was 30-280 μM with a response time of 10 sec. PMID:26504954

  16. On the oxygen reactivity of flavoprotein oxidases: an oxygen access tunnel and gate in brevibacterium sterolicum cholesterol oxidase.

    PubMed

    Piubelli, Luciano; Pedotti, Mattia; Molla, Gianluca; Feindler-Boeckh, Susanne; Ghisla, Sandro; Pilone, Mirella S; Pollegioni, Loredano

    2008-09-01

    The flavoprotein cholesterol oxidase from Brevibacterium sterolicum (BCO) possesses a narrow channel that links the active center containing the flavin to the outside solvent. This channel has been proposed to serve for the access of dioxygen; it contains at its "bottom" a Glu-Arg pair (Glu-475-Arg-477) that was found by crystallographic studies to exist in two forms named "open" and "closed," which in turn was suggested to constitute a gate functioning in the control of oxygen access. Most mutations of residues that flank the channel have minor effects on the oxygen reactivity. Mutations of Glu-311, however, cause a switch in the basic kinetic mechanism of the reaction of reduced BCO with dioxygen; wild-type BCO and most mutants show a saturation behavior with increasing oxygen concentration, whereas for Glu-311 mutants a linear dependence is found that is assumed to reflect a "simple" second order process. This is taken as support for the assumption that residue Glu-311 finely tunes the Glu-475-Arg-477 pair, forming a gate that functions in modulating the access/reactivity of dioxygen.

  17. Reusable and Mediator-Free Cholesterol Biosensor Based on Cholesterol Oxidase Immobilized onto TGA-SAM Modified Smart Bio-Chips

    PubMed Central

    Rahman, Mohammed M.

    2014-01-01

    A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields. PMID:24949733

  18. Enzymatic activity of cholesterol oxidase immobilized onto polymer nanoparticles mediated by Congo red.

    PubMed

    Silva, Rubens A; Carmona-Ribeiro, Ana Maria; Petri, Denise F S

    2013-10-01

    Poly(ethylene glycol), PEG, decorated polystyrene (PS) nanoparticles were synthesized and characterized by means of dynamic light scattering (DLS), zeta (ζ) potential measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption of Congo red (CR) onto PS/PEG particles was evidenced by the decrease of ζ potential values and increase in the particles mean diameter in comparison to bare particles. Cholesterol oxidase (ChOx), the main enzyme in the oxidation of cholesterol, adsorbed onto PS/PEG and PS/PEG/CR particles, as revealed by the increase in the particles mean size and spectrophotometry. The enzymatic activity of free and immobilized ChOx was determined as a function of time by means of a coupled reaction with horseradish peroxidase. The activity of free ChOx decreased with time, while the activity of immobilized ChOx increased with time; after 1h reaction the latter was half of the former. Freeze-drying the ChOx covered PS/PEG/CR particles allowed their storage for at least one month under room conditions without loss of enzymatic activity. Conjugation effects between CR and ChOx or cholesterol evidenced by circular dichroism and spectrophotometry rendered a conformational state of ChOx, such that the enzymatic action was favored. ChOx adsorbed onto PS/PEG presents no enzymatic activity, probably due to ChOx denaturation or unfavorable orientation. Freeze-dried and freshly prepared dispersions of ChOx immobilized onto PS/PEG/CR particles yielded linear response in the cholesterol concentration range of 100mgdL(-1) (lowest limit of normal blood concentration) to 300mgdL(-1) (high risk level).

  19. The physiologic role of alternative oxidase in Ustilago maydis.

    PubMed

    Juárez, Oscar; Guerra, Guadalupe; Velázquez, Isabel; Flores-Herrera, Oscar; Rivera-Pérez, R E; Pardo, Juan P

    2006-10-01

    Alternative oxidase (AOX) is a ubiquitous respiratory enzyme found in plants, fungi, protists and some bacterial species. One of the major questions about this enzyme is related to its metabolic role(s) in cellular physiology, due to its capacity to bypass the proton-pumping cytochrome pathway, and as a consequence it has great energy-wasting potential. In this study, the physiological role and regulatory mechanisms of AOX in the fungal phytopathogen Ustilago maydis were studied. We found evidence for at least two metabolic functions for AOX in this organism, as a major part of the oxidative stress-handling machinery, a well-described issue, and as part of the mechanisms that increase the metabolic plasticity of the cell, a role that might be valuable for organisms exposed to variations in temperature, nutrient source and availability, and biotic or abiotic factors that limit the activity of the cytochrome pathway. Experiments under different culture conditions of ecological significance for this organism revealed that AOX activity is modified by the growth stage of the culture, amino acid availability and growth temperature. In addition, nucleotide content, stimulation of AOX by AMP and respiratory rates obtained after inhibition of the cytochrome pathway showed that fungal/protist AOX is activated under low-energy conditions, in contrast to plant AOX, which is activated under high-energy conditions. An estimation of the contribution of AOX to cell respiration was performed by comparing the steady-state concentration of adenine nucleotides, the mitochondrial membrane potential, and the respiratory rate.

  20. Colorimetric Quantification of Glucose and Cholesterol in Human Blood Using a Nanocomposite Entrapping Magnetic Nanoparticles and Oxidases.

    PubMed

    Kim, Moon Il; Cho, Daeyeon; Park, Hyun Gyu

    2015-10-01

    In this study, a microscale well-plate colorimetric assay for the multiplexed detection of glucose and cholesterol in clinical human blood samples has been developed. This system utilized one-pot nanocomposite entrapping Fe3O4 magnetic nanoparticles (MNPs) as peroxidase mimetics and glucose oxidase (GOx)/cholesterol oxidase (ChOx) in mesoporous silica to detect glucose and cholesterol in blood samples. The sensing mechanism involves the generation of H2O2 by the catalytic action of an immobilized oxidase on the target molecules in the sample. This subsequently activates the MNPs in the mesopores, thereby leading to the conversion of the substrate into a colored end product. This strategy is used to detect the target glucose or cholesterol molecules in the concentration range of 15-250 mg/dL. The response is highly linear and the lower detection limit is 7.5 mg/dL. The aforementioned colorimetric assay is extremely convenient, and it exhibits a high degree of linearity, precision, and reproducibility when employing real human blood samples. Therefore, this assay can be used in clinical practice for the multiplexed and reliable quantification of glucose and cholesterol. PMID:26726446

  1. Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.

    PubMed

    Kumari, Lata; Kanwar, Shamsher S

    2016-01-01

    A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 μmol min ml(-1), respectively. The purified COX showed very little cytotoxicity associated with it. PMID:26453032

  2. Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode.

    PubMed

    Gholivand, Mohammad Bagher; Khodadadian, Mehdi

    2014-03-15

    Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR-IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163 mA mM(-1)cm(-2), a response time less than 6s, and a linear range of 0.25-215 μM (R(2)>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis-Menten constant (KM(app)) was calculated to be 2.32 mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDs<5%) with minimal interference from the coexisting electroactive compounds such as ascorbic acid and uric acid. The CAT/ChOx/GR-IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples.

  3. Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

    PubMed

    Pathak, Lakshmi; Singh, Vineeta; Niwas, Ram; Osama, Khwaja; Khan, Saif; Haque, Shafiul; Tripathi, C K M; Mishra, B N

    2015-01-01

    Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.

  4. Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi.

    PubMed Central

    Machang'u, R S; Prescott, J F

    1991-01-01

    Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 2. Fig. 5. PMID:1790488

  5. Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

    PubMed Central

    Niwas, Ram; Osama, Khwaja; Khan, Saif; Haque, Shafiul; Tripathi, C. K. M.; Mishra, B. N.

    2015-01-01

    Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO4, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500. PMID:26368924

  6. Physiological role of alternative oxidase (from yeasts to plants).

    PubMed

    Rogov, A G; Zvyagilskaya, R A

    2015-04-01

    Mitochondria of all so far studied organisms, with the exception of Archaea, mammals, some yeasts, and protists, contain, along with the classical phosphorylating cytochrome pathway, a so-called cyanide-insensitive alternative oxidase (AOX) localized on the matrix side of the mitochondrial inner membrane, and electron transport through which is not coupled with ATP synthesis and energy accumulation. Mechanisms underlying plentiful functions of AOX in organisms at various levels of organization ranging from yeasts to plants are considered. First and foremost, AOX provides a chance of cell survival after inhibiting the terminal components of the main respiratory chain or losing the ability to synthesize these components. The vitally important role of AOX is obvious in thermogenesis of thermogenic plant organs where it becomes the only terminal oxidase with a very high activity, and the energy of substrate oxidation by this respiratory pathway is converted into heat, thus promoting evaporation of volatile substances attracting pollinating insects. AOX plays a fundamentally significant role in alleviating or preventing oxidative stress, thus ensuring the defense against a wide range of stresses and adverse environmental conditions, such as changes in temperature and light intensities, osmotic stress, drought, and attack by incompatible strains of bacterial pathogens, phytopathogens, or their elicitors. Participation of AOX in pathogen survival during its existence inside the host, in antivirus defense, as well as in metabolic rearrangements in plants during embryogenesis and cell differentiation is described. Examples are given to demonstrate that AOX might be an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals.

  7. Physiological role of alternative oxidase (from yeasts to plants).

    PubMed

    Rogov, A G; Zvyagilskaya, R A

    2015-04-01

    Mitochondria of all so far studied organisms, with the exception of Archaea, mammals, some yeasts, and protists, contain, along with the classical phosphorylating cytochrome pathway, a so-called cyanide-insensitive alternative oxidase (AOX) localized on the matrix side of the mitochondrial inner membrane, and electron transport through which is not coupled with ATP synthesis and energy accumulation. Mechanisms underlying plentiful functions of AOX in organisms at various levels of organization ranging from yeasts to plants are considered. First and foremost, AOX provides a chance of cell survival after inhibiting the terminal components of the main respiratory chain or losing the ability to synthesize these components. The vitally important role of AOX is obvious in thermogenesis of thermogenic plant organs where it becomes the only terminal oxidase with a very high activity, and the energy of substrate oxidation by this respiratory pathway is converted into heat, thus promoting evaporation of volatile substances attracting pollinating insects. AOX plays a fundamentally significant role in alleviating or preventing oxidative stress, thus ensuring the defense against a wide range of stresses and adverse environmental conditions, such as changes in temperature and light intensities, osmotic stress, drought, and attack by incompatible strains of bacterial pathogens, phytopathogens, or their elicitors. Participation of AOX in pathogen survival during its existence inside the host, in antivirus defense, as well as in metabolic rearrangements in plants during embryogenesis and cell differentiation is described. Examples are given to demonstrate that AOX might be an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals. PMID:25869356

  8. A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase

    SciTech Connect

    Lyubimov, Artem Y.; Chen, Lin; Sampson, Nicole S.; Vrielink, Alice

    2009-11-01

    The importance of active-site electrostatics for oxidative and reductive half-reactions in a redox flavoenzyme (cholesterol oxidase) have been investigated by a combination of biochemistry and atomic resolution crystallography. A detailed examination of active-site dynamics demonstrates that the oxidation of substrate and the re-oxidation of the flavin cofactor by molecular oxygen are linked by a single active-site asparagine. Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3β-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH⋯π interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall k{sub cat}. Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH⋯π interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered.

  9. Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization.

    PubMed

    Torabi, Seyed-Fakhreddin; Khajeh, Khosro; Ghasempur, Salehe; Ghaemi, Nasser; Siadat, Seyed-Omid Ranaei

    2007-08-31

    In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.

  10. Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity.

    PubMed

    Granzotto, A; Suwalsky, M; Zatta, P

    2011-08-01

    Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection. PMID:21726769

  11. Finding New Enzymes from Bacterial Physiology: A Successful Approach Illustrated by the Detection of Novel Oxidases in Marinomonas mediterranea

    PubMed Central

    Sanchez-Amat, Antonio; Solano, Francisco; Lucas-Elío, Patricia

    2010-01-01

    The identification and study of marine microorganisms with unique physiological traits can be a very powerful tool discovering novel enzymes of possible biotechnological interest. This approach can complement the enormous amount of data concerning gene diversity in marine environments offered by metagenomic analysis, and can help to place the activities associated with those sequences in the context of microbial cellular metabolism and physiology. Accordingly, the detection and isolation of microorganisms that may be a good source of enzymes is of great importance. Marinomonas mediterranea, for example, has proven to be one such useful microorganism. This Gram-negative marine bacterium was first selected because of the unusually high amounts of melanins synthesized in media containing the amino acid l-tyrosine. The study of its molecular biology has allowed the cloning of several genes encoding oxidases of biotechnological interest, particularly in white and red biotechnology. Characterization of the operon encoding the tyrosinase responsible for melanin synthesis revealed that a second gene in that operon encodes a protein, PpoB2, which is involved in copper transfer to tyrosinase. This finding made PpoB2 the first protein in the COG5486 group to which a physiological role has been assigned. Another enzyme of interest described in M. mediterranea is a multicopper oxidase encoding a membrane-associated enzyme that shows oxidative activity on a wide range of substrates typical of both laccases and tyrosinases. Finally, an enzyme very specific for l-lysine, which oxidises this amino acid in epsilon position and that has received a new EC number (1.4.3.20), has also been described for M. mediterranea. Overall, the studies carried out on this bacterium illustrate the power of exploring the physiology of selected microorganisms to discover novel enzymes of biotechnological relevance. PMID:20411113

  12. [A history and review of cholesterol ester transfer protein inhibitors and their contribution to the understanding of the physiology and pathophysiology of high density lipoprotein].

    PubMed

    Corral, Pablo; Schreier, Laura

    2014-01-01

    There is irrefutable evidence that statins reduce the risk of cardiovascular events in a magnitude proportional to the intensity of the decrease in cholesterol transport by the low density lipoproteins. Despite this great advance there is still a residual risk of cardiovascular events. For this reason, an increase in the levels of high density lipoprotein is considered in order to boost the main action of this lipoprotein, which is reverse cholesterol transport. Distinct classes of evidence (epidemiological, genetic, and pathophysiological) show that the inhibition and/or modulation of cholesterol ester transfer protein increases plasma high density lipoprotein-cholesterol levels. The main reason for presenting this review is to look at the physiology of cholesterol ester transfer protein, its interrelationship with high density lipoproteins, and to give an update on the development of different cholesterol ester transfer protein inhibitor/modulator molecules. PMID:24094503

  13. [A history and review of cholesterol ester transfer protein inhibitors and their contribution to the understanding of the physiology and pathophysiology of high density lipoprotein].

    PubMed

    Corral, Pablo; Schreier, Laura

    2014-01-01

    There is irrefutable evidence that statins reduce the risk of cardiovascular events in a magnitude proportional to the intensity of the decrease in cholesterol transport by the low density lipoproteins. Despite this great advance there is still a residual risk of cardiovascular events. For this reason, an increase in the levels of high density lipoprotein is considered in order to boost the main action of this lipoprotein, which is reverse cholesterol transport. Distinct classes of evidence (epidemiological, genetic, and pathophysiological) show that the inhibition and/or modulation of cholesterol ester transfer protein increases plasma high density lipoprotein-cholesterol levels. The main reason for presenting this review is to look at the physiology of cholesterol ester transfer protein, its interrelationship with high density lipoproteins, and to give an update on the development of different cholesterol ester transfer protein inhibitor/modulator molecules.

  14. Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability.

    PubMed

    Kasabe, Pramod J; Mali, Geetanjali T; Dandge, Padma B

    2015-12-01

    The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the 'Chilika Lake' located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3β-hydroxysteroids with Km of 1.1 × 10(-4)M for cholesterol. The pH, 8.0 and the temperature, 37 °C were required for its optimum activity. Enzyme is considerably stable at pH 6.0-8.5 and temperature up to 50 °C. At 4 and 30 °C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn(2+), Cu(2+), Ag+, Fe(3+), Ba(2+) inhibited the enzyme activity >60% while Hg(2+) served a potent inhibitor whereas Mg(2+) found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications.

  15. Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability.

    PubMed

    Kasabe, Pramod J; Mali, Geetanjali T; Dandge, Padma B

    2015-12-01

    The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the 'Chilika Lake' located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3β-hydroxysteroids with Km of 1.1 × 10(-4)M for cholesterol. The pH, 8.0 and the temperature, 37 °C were required for its optimum activity. Enzyme is considerably stable at pH 6.0-8.5 and temperature up to 50 °C. At 4 and 30 °C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn(2+), Cu(2+), Ag+, Fe(3+), Ba(2+) inhibited the enzyme activity >60% while Hg(2+) served a potent inhibitor whereas Mg(2+) found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications. PMID:26276474

  16. Production, Purification, and Identification of Cholest-4-en-3-one Produced by Cholesterol Oxidase from Rhodococcus sp. in Aqueous/Organic Biphasic System.

    PubMed

    Wu, Ke; Li, Wei; Song, Jianrui; Li, Tao

    2015-01-01

    Cholest-4-en-3-one has positive uses against obesity, liver disease, and keratinization. It can be applied in the synthesis of steroid drugs as well. Most related studies are focused on preparation of cholest-4-en-3-one by using whole cells as catalysts, but production of high-quality cholest-4-en-3-one directly from cholesterol oxidase (COD) using an aqueous/organic two-phase system has been rarely explored. This study set up an enzymatic reaction system to produce cholest-4-en-3-one. We developed and optimized the enzymatic reaction system using COD from COX5-6 (a strain of Rhodococcus) instead of whole-cell biocatalyst. This not only simplifies and accelerates the production but also benefits the subsequent separation and purification process. Through extraction, washing, evaporation, column chromatography, and recrystallization, we got cholest-4-en-3-one with purity of 99.78%, which was identified by nuclear magnetic resonance, mass spectroscopy, and infrared spectroscopy. In addition, this optimized process of cholest-4-en-3-one production and purification can be easily scaled up for industrial production, which can largely decrease the cost and guarantee the purity of the product.

  17. Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid

    PubMed Central

    Miller, Norman E.; Olszewski, Waldemar L.; Miller, Irina P.; Nanjee, Mahmud N.

    2016-01-01

    Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins. PMID:27471469

  18. Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid.

    PubMed

    Miller, Norman E; Olszewski, Waldemar L; Miller, Irina P; Nanjee, Mahmud N

    2016-01-01

    Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins. PMID:27471469

  19. Membrane cholesterol modulates galanin-GalR2 interaction.

    PubMed

    Pang, L; Graziano, M; Wang, S

    1999-09-14

    The neuropeptide galanin mediates a number of diverse physiological and pathophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expressed in Chinese hamster ovary (CHO) cells was examined. Reduction of membrane cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by culturing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-depleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n >/= 3) in a positively cooperative manner. This interaction appears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue galanin binding. The inability of some of these analogues to rescue the binding activity also suggests that binding of galanin to GalR2 is independent of membrane fluidity as, like cholesterol, cholesterol analogues generally rigidize membranes. In addition, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, treatment of membranes, with filipin, a molecule that clusters cholesterol within the membranes, or with cholesterol oxidase resulted in markedly reduced galanin binding. Incubation of membranes with 100 microM GTP-gamma-S did not alter the IC(50) for CD in the prebinding assay treatment suggesting that the effect of cholesterol was independent of G protein interaction. Preincubation of intact cells with CD also drastically impaired the ability of galanin to activate intracellular inositol phosphate accumulation in GalR2-transfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of Gal

  20. Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

    PubMed Central

    Neuvonen, Maarit; Manna, Moutusi; Mokkila, Sini; Javanainen, Matti; Rog, Tomasz; Liu, Zheng; Bittman, Robert; Vattulainen, Ilpo; Ikonen, Elina

    2014-01-01

    Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone. PMID:25157633

  1. Cost-effective and highly sensitive cholesterol microsensors with fast response based on the enzyme-induced conductivity change of polyaniline

    NASA Astrophysics Data System (ADS)

    Fang, Kuan-Chung; Chu, Chia-Ho; Hsu, Chen-Pin; Kang, Yen-Wen; Fang, Jung-Ying; Hsu, Chia-Hsien; Huang, Yu-Fen; Chen, Chih-Chen; Li, Sheng-Shian; Andrew Yeh, J.; Yao, Da-Jeng; Wang, Yu-Lin

    2014-09-01

    In this study, a cost-effective and highly sensitive cholesterol microsensor, which is consisted of cholesterol oxidase (ChOx), horseradish peroxidase (HRP), and polyaniline (PANI), was developed based on the enzyme-induced conductivity change of PANI with fast response. Hydrogen peroxide is produced via the reaction between cholesterol and ChOx, which was immobilized in a dialysis membrane. The produced hydrogen peroxide can oxidize HRP, which can be reduced by oxidizing PANI, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be oxidized again by hydrogen peroxide and the cycle of the oxidation/reduction continues until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecules. Cholesterol was detected near the physiological concentrations ranging from 100 mg/dl to 400 mg/dl with the cholesterol microsensors. The results show linear relation between cholesterol concentration and the conductivity change of the PANI. The microsensor showed no response to cholesterol when the PANI was standalone without cholesterol oxidase immobilized, indicating that the enzymatic reaction is required for cholesterol detection. The simple process of the sensor fabrication allows the sensor to be cost-effective and disposable usage. This electronic cholesterol microsensor is promising for point-of-care health monitoring in cholesterol level with low cost and fast response.

  2. Cost-effective and highly sensitive cholesterol microsensors with fast response based on the enzyme-induced conductivity change of polyaniline

    SciTech Connect

    Fang, Kuan-Chung; Chu, Chia-Ho; Hsu, Chen-Pin; Kang, Yen-Wen; Fang, Jung-Ying; Chen, Chih-Chen; Li, Sheng-Shian; Andrew Yeh, J.; Yao, Da-Jeng; Wang, Yu-Lin; Hsu, Chia-Hsien; Huang, Yu-Fen

    2014-09-15

    In this study, a cost-effective and highly sensitive cholesterol microsensor, which is consisted of cholesterol oxidase (ChOx), horseradish peroxidase (HRP), and polyaniline (PANI), was developed based on the enzyme-induced conductivity change of PANI with fast response. Hydrogen peroxide is produced via the reaction between cholesterol and ChOx, which was immobilized in a dialysis membrane. The produced hydrogen peroxide can oxidize HRP, which can be reduced by oxidizing PANI, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be oxidized again by hydrogen peroxide and the cycle of the oxidation/reduction continues until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecules. Cholesterol was detected near the physiological concentrations ranging from 100 mg/dl to 400 mg/dl with the cholesterol microsensors. The results show linear relation between cholesterol concentration and the conductivity change of the PANI. The microsensor showed no response to cholesterol when the PANI was standalone without cholesterol oxidase immobilized, indicating that the enzymatic reaction is required for cholesterol detection. The simple process of the sensor fabrication allows the sensor to be cost-effective and disposable usage. This electronic cholesterol microsensor is promising for point-of-care health monitoring in cholesterol level with low cost and fast response.

  3. What's Cholesterol?

    MedlinePlus

    ... Most cholesterol is LDL (low-density lipoprotein) cholesterol. LDL cholesterol is more likely to clog blood vessels because ... Here's a way to remember the difference: the LDL cholesterol is the bad kind, so call it "lousy" ...

  4. Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

    PubMed

    Han, Hai; Miyoshi, Yurika; Koga, Reiko; Mita, Masashi; Konno, Ryuichi; Hamase, Kenji

    2015-12-10

    D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

  5. Impact of physiological levels of chenodeoxycholic acid supplementation on intestinal and hepatic bile acid and cholesterol metabolism in Cyp7a1-deficient mice

    PubMed Central

    Jones, Ryan D.; Lopez, Adam M.; Tong, Ernest Y.; Posey, Kenneth S.; Chuang, Jen-Chieh; Repa, Joyce J.; Turley, Stephen D.

    2014-01-01

    Mice deficient in cholesterol 7α-hydroxylase (Cyp7a1) have a diminished bile acid pool (BAP) and therefore represent a useful model for investigating the metabolic effects of restoring the pool with a specific BA. Previously we carried out such studies in Cyp7a1−/−mice fed physiological levels of cholic acid (CA) and achieved BAP restoration, along with an increased CA enrichment, at a dietary level of just 0.03% (w/w). Here we demonstrate that in Cyp7a1−/− mice fed chenodeoxycholic acid (CDCA) at a level of 0.06 % (w/w), the BAP was restored to normal size and became substantially enriched with muricholic acid (MCA)(>70%), leaving the combined contribution of CA and CDCA to be <15%. This resulted in a partial to complete reversal of the main changes in cholesterol and BA metabolism associated with Cyp7a1 deficiency such as an elevated rate of intestinal sterol synthesis, an enhanced level of mRNA for Cyp8b1 in the liver, and depressed mRNA levels for Ibabp, Shp and Fgf15 in the distal small intestine. When Cyp7a1−/− and matching Cyp7a1+/+ mice were fed a diet with added cholesterol (0.2%) (w/w), either alone, or also containing CDCA (0.06%) (w/w) or CA (0.03%) (w/w) for 18 days, the hepatic total cholesterol concentrations (mg/g) in the Cyp7a1−/− mice were 26.9±3.7, 16.4±0.9 and 47.6±1.9, respectively, vs 4.9±0.4, 5.0±0.7 and 6.4±1.9, respectively in the corresponding Cyp7a1+/+ controls. These data affirm the importance of using moderate levels of dietary BA supplementation to elicit changes in hepatic cholesterol metabolism through shifts in BAP size and composition. PMID:25447797

  6. Lipoprotein(a) particle concentration and lipoprotein(a) cholesterol assays yield discordant classification of patients into four physiologically discrete groups.

    PubMed

    Konerman, Matthew; Kulkarni, Krishnaji; Toth, Peter P; Jones, Steven R

    2012-01-01

    There is little known about the relative predictive value of different lipoprotein(a) [Lp(a)] assays in clinical use, although each has been shown to predict similar incremental risk over conventional clinical and lipid risk factors. Thus, we examined the classification behavior of two commonly used Lp(a) assays and their associations with other lipid parameters. Serum lipid and Lp(a) concentrations were measured in 144 primary and secondary prevention patients. Lp(a) cholesterol [Lp(a)-C] was measured with the Vertical Auto Profile (upper limit of normal, 10 mg/dL). Lp(a) particle concentrations [Lp(a)-P] were measured with an isoform-independent molar assay (upper limit of normal, 70 nmol/L). The subjects were divided into the following four groups on the basis of their Lp(a)-C and Lp(a)-P levels: normal Lp(a)-P and Lp(a)-C; high Lp(a)-P and normal Lp(a)-C; normal Lp(a)-P and high Lp(a)-C; and high Lp(a)-P and Lp(a)-C. The proportion of subjects with values above the upper limit of normal was similar with both assays (P = .15). However, the Lp(a)-C and Lp(a)-P assays discordantly classified 23% of the study's subjects. In addition, the four Lp(a)-defined groups displayed differences in their relationships with other lipoproteins. The two groups with elevated Lp(a)-C showed significant associations with higher high-density lipoprotein cholesterol, apolipoprotein AI, and high-density lipoprotein cholesterol/apolipoprotein AI ratios. Triglycerides were also noted to be above normal in discordant and normal within concordant Lp(a) groups. Finally, the amount of cholesterol per Lp(a) particle [Lp(a)-C/Lp(a)-P] varied widely across the four groups. These findings suggest that the four Lp(a)-defined groups are physiologically discrete. Further investigation is warranted to assess which parameters among Lp(a)-P, Lp(a)-C, and Lp(a)-C/Lp(a)-P can be used to more accurately characterize Lp(a)-associated cardiovascular risk. PMID:22836074

  7. Development of mediator-type biosensor to wirelessly monitor whole cholesterol concentration in fish.

    PubMed

    Takase, Mai; Murata, Masataka; Hibi, Kyoko; Huifeng, Ren; Endo, Hideaki

    2014-04-01

    We developed a wireless monitoring system to monitor fish condition by tracking the change in whole cholesterol concentration. The whole cholesterol concentration of fish is a source of steroid hormones or indicator of immunity level, which makes its detection important for tracking physiological condition of fish. Wireless monitoring system comprises of mediator-type biosensor and wireless transmission device. Biosensor is implantable to fish body, and transmission device is so light, in that fish is allowed to swim freely during monitoring. Cholesterol esterase and oxidase were fixated on to the detection site of biosensor and used to detect the whole cholesterol concentration. However, cholesterol oxidase incorporates oxidation-reduction reaction of oxygen for detection, which concentration fluctuates easily due to change in environmental condition. Meanwhile, mediator-type biosensor enables monitoring of whole cholesterol concentration by using mediator to substitute that oxidation-reduction reaction of oxygen. Characteristic of fabricated mediator-type biosensor was tested. The sensor output current of mediator-type biosensor remained stable compared to output current of non-mediator-type biosensor under fluctuating oxygen concentration of 0-8 ppm, which implied that this sensor is less affected by change in dissolved oxygen concentration. That biosensor was then implanted into fish for wireless monitoring. As a result, approximately 48 h of real-time monitoring was successful.

  8. About Cholesterol

    MedlinePlus

    ... High Blood Pressure Tools & Resources Stroke More About Cholesterol Updated:Aug 10,2016 It may surprise you ... our bodies to keep us healthy. What is cholesterol and where does it come from? Cholesterol is ...

  9. Physiology

    ERIC Educational Resources Information Center

    Kay, Ian

    2008-01-01

    Underlying recent developments in health care and new treatments for disease are advances in basic medical sciences. This edition of "Webwatch" focuses on sites dealing with basic medical sciences, with particular attention given to physiology. There is a vast amount of information on the web related to physiology. The sites that are included here…

  10. Localization of cholesterol in sphingomyelinase-treated fibroblasts.

    PubMed Central

    Pörn, M I; Slotte, J P

    1995-01-01

    The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible

  11. Localization of cholesterol in sphingomyelinase-treated fibroblasts.

    PubMed

    Pörn, M I; Slotte, J P

    1995-05-15

    The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible

  12. Physiological and biochemical characterisation of watered and drought-stressed barley mutants in the HvDWARF gene encoding C6-oxidase involved in brassinosteroid biosynthesis.

    PubMed

    Janeczko, Anna; Gruszka, Damian; Pociecha, Ewa; Dziurka, Michał; Filek, Maria; Jurczyk, Barbara; Kalaji, Hazem M; Kocurek, Maciej; Waligórski, Piotr

    2016-02-01

    Brassinosteroids (BR) are plant steroid hormones that were discovered more than thirty years ago, but their physiological function has yet to be fully explained. The aim of the study was to answer the question of whether/how disturbances in the production of BR in barley affects the plant's metabolism and development under conditions of optimal watering and drought. Mutants with an impaired production of BR are one of the best tools in research aimed at understanding the mechanisms of action of these hormones. The study used barley cultivars with a normal BR synthesis (wild type) and semi-dwarf allelic mutants with an impaired activity of C6-oxidase (mutation in HvDWARF), which resulted in a decreased BR synthesis. Half of the plants were subjected to drought stress in the seedling stage and the other half were watered optimally. Plants with impaired BR production were characterised by a lower height and developmental retardation. Under both optimal watering and drought, BR synthesis disorders caused the reduced production of ABA and cytokinins, but not auxins. The BR mutants also produced less osmoprotectant (proline). The optimally watered and drought-stressed mutants accumulated less sucrose, which was accompanied by changes in the production of other soluble sugars. The increased content of fructooligosaccharide (kestose) in optimally watered mutants would suggest that BR is a negative regulator of kestose production. The decreased level of nystose in the drought-stressed mutants also suggests BR involvement in the regulation of the production of this fructooligosaccharide. The accumulation of the transcripts of genes associated with stress response (hsp90) was lower in the watered and drought-stressed BR-deficient mutants. In turn, the lower efficiency of photosystem II and the net photosynthetic rate in mutants was revealed only under drought conditions. The presented research allows for the physiological and biochemical traits of two BR-barley mutants to be

  13. Cholesterol transport in model membranes

    NASA Astrophysics Data System (ADS)

    Garg, Sumit; Porcar, Lionel; Butler, Paul; Perez-Salas, Ursula

    2010-03-01

    Physiological processes distribute cholesterol unevenly within the cell. The levels of cholesterol are maintained by intracellular transport and a disruption in the cell's ability to keep these normal levels will lead to disease. Exchange rates of cholesterol are generally studied in model systems using labeled lipid vesicles. Initially donor vesicles have all the cholesterol and acceptor vesicles are devoid of it. They are mixed and after some time the vesicles are separated and cholesterol is traced in each vesicle. The studies performed up to date have significant scatter indicating that the methodologies are not consistent. The present work shows in-situ Time-Resolved SANS studies of cholesterol exchange rates in unsaturated PC lipid vesicles. Molecular dynamics simulations were done to investigate the energetic and kinetic behavior of cholesterol in this system. This synergistic approach will provide insight into our efforts to understand cholesterol traffic.

  14. Cholesterol (image)

    MedlinePlus

    Cholesterol is a soft, waxy substance that is present in all parts of the body including the ... and obtained from animal products in the diet. Cholesterol is manufactured in the liver and is needed ...

  15. Cholesterol determination using protein-templated fluorescent gold nanocluster probes.

    PubMed

    Chen, Xi; Baker, Gary A

    2013-11-12

    We describe the development of a fluorescent biosensor platform for soluble cholesterol based on bovine serum albumin-stabilized gold nanocluster probes co-dissolved with cholesterol oxidase (ChOx) in a surfactant emulsion system. Selective enzymatic oxidation of cholesterol to cholest-4-en-3-one by ChOx produces stoichiometric amounts of H2O2 by-product, generating a quenching response signaling the presence of cholesterol at clinically relevant levels (LOD ∼12 μM).

  16. Cholesterol depletion induces autophagy

    SciTech Connect

    Cheng, Jinglei; Ohsaki, Yuki; Tauchi-Sato, Kumi; Fujita, Akikazu; Fujimoto, Toyoshi . E-mail: tfujimot@med.nagoya-u.ac.jp

    2006-12-08

    Autophagy is a mechanism to digest cells' own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5 mM methyl-{beta}-cyclodextrin or 10-20 {mu}g/ml nystatin for 1 h, or metabolically by 20 {mu}M mevastatin and 200 {mu}M mevalonolactone along with 10% lipoprotein-deficient serum for 2-3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks' solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50 {mu}m in length. The increase of LC3-II by methyl-{beta}-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

  17. [Alternative oxidase in industrial fungi].

    PubMed

    Gu, Shuai; Liu, Qiang; He, Hao; Li, Shuang

    2015-01-01

    Filamentous fungi have been used in industrial fermentation extensively. Based on non-phosphorylating electron transport process, alternative respiration pathway (ARP) acts as an energy overflow, which can balance carbon metabolism and electron transport, allow the continuance of tricarboxylic acid cycle without the formation of ATP, and permit the turnover of carbon skeletons. Alternative respiration pathway also plays an important role in the stress response of fungi and the physiological function of conditioned pathogen. Alternative oxidase (AOX) is the terminal oxidase responsible for the activity of alternative respiration pathway, which exists widely in higher plants, parts of fungi and algae. Owing to the property that alternative oxidase (AOX) is sensitive to salicylhydroxamic acid (SHAM) and insensitive to conventional inhibitors of cytochrome respiration, alternative respiration pathway by AOX is also named as cyanide-resistant respiration (CRR). In recent years, the study of the alternative respiration pathway and alternative oxidase has been a hot topic in the area involving cellular respiration metabolism. In this review we summarized the latest research advances about the functions of alternative respiration pathway and alternative oxidase in industrial fungi.

  18. Women and Cholesterol

    MedlinePlus

    ... Blood Pressure Tools & Resources Stroke More Women and Cholesterol Updated:Apr 1,2016 The female sex hormone ... Glossary Related Sites Nutrition Center My Life Check Cholesterol • Home • About Cholesterol • Why Cholesterol Matters • Understand Your ...

  19. Cholesterol IQ Quiz

    MedlinePlus

    ... Pressure High Blood Pressure Tools & Resources Stroke More Cholesterol IQ Quiz Updated:Feb 2,2015 Begin the quiz Cholesterol • Home • About Cholesterol Introduction Good vs. Bad Cholesterol ...

  20. NADPH Oxidase and Neurodegeneration

    PubMed Central

    Hernandes, Marina S; Britto, Luiz R G

    2012-01-01

    NADPH oxidase (Nox) is a unique, multi-protein, electron transport system that produces large amounts of superoxide via the reduction of molecular oxygen. Nox-derived reactive oxygen species (ROS) are known to be involved in a variety of physiological processes, including host defense and signal transduction. However, over the past decade, the involvement of (Nox)-dependent oxidative stress in the pathophysiology of several neurodegenerative diseases has been increasingly recognized. ROS produced by Nox proteins contribute to neurodegenerative diseases through distinct mechanisms, such as oxidation of DNA, proteins, lipids, amino acids and metals, in addition to activation of redox-sensitive signaling pathways. In this review, we discuss the recent literature on Nox involvement in neurodegeneration, focusing on Parkinson and Alzheimer diseases. PMID:23730256

  1. Physiological responses and endogenous cytokinin profiles of tissue-cultured 'Williams' bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments.

    PubMed

    Aremu, Adeyemi O; Bairu, Michael W; Novák, Ondřej; Plačková, Lenka; Zatloukal, Marek; Doležal, Karel; Finnie, Jeffrey F; Strnad, Miroslav; Van Staden, Johannes

    2012-12-01

    The effect of supplementing either meta-topolin (mT) or N(6)-benzyladenine (BA) requiring cultures with roscovitine (6-benzylamino-2-[1(R)-(hydroxymethyl)propyl]amino-9-isopropylpurine), a cyclin-dependent kinase (CDK) and N-glucosylation inhibitor, and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine), an inhibitor of cytokinin (CK) degradation, on the endogenous CK profiles and physiology of banana in vitro was investigated. Growth parameters including multiplication rate and biomass were recorded after 42 days. Endogenous CK levels were quantified using UPLC-MS/MS while the photosynthetic pigment and phenolic contents were evaluated spectrophotometrically. The highest regeneration rate (93 %) was observed in BA + roscovitine while mT + INCYDE plantlets produced most shoots. Treatment with BA + roscovitine had the highest shoot length and biomass. Although not significant, there was a higher proanthocyanidin level in BA + roscovitine treatments compared to the control (BA). The levels of total phenolics and flavonoids were significantly higher in mT + roscovitine treatment than in the mT-treated regenerants. The presence of roscovitine and/or INCYDE had no significant effect on the photosynthetic pigments of the banana plantlets. Forty-seven aromatic and isoprenoid CKs categorized into nine CK-types were detected at varying concentrations. The presence of mT + roscovitine and/or INCYDE increased the levels of O-glucosides while 9-glucosides were higher in the presence of BA. Generally, the underground parts had higher CK levels than the aerial parts; however, the presence of INCYDE increased the level of CK quantified in the aerial parts. From a practical perspective, the use of roscovitine and INCYDE in micropropagation could be crucial in the alleviation of commonly observed in vitro-induced physiological abnormalities.

  2. Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis.

    PubMed

    Honsho, Masanori; Abe, Yuichi; Fujiki, Yukio

    2015-11-27

    Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

  3. [Basic mechanisms: absorption and excretion of cholesterol and other sterols].

    PubMed

    Cofan Pujol, Montserrat

    2014-01-01

    Cholesterol is of vital importance for vertebrate cell membrane structure and function. It is obvious that adequate regulation of cholesterol homeostasis is essential. Hypercholesterolemia promotes atherosclerosis and thereby represents a major risk factor for cardiovascular disease. The liver has been considered the major site of control in maintenance of cholesterol homeostasis. The liver facilitates clearance of (very) low density lipoprotein particles and cholesterol-containing chylomicron remnants, synthesizes cholesterol, synthesizes and secretes (nascent) high density lipoprotein particles, secretes cholesterol and bile salts to bile, and is involved in reverse cholesterol transport. In recent years, however, the importance of the intestine in many aspects of cholesterol physiology is increasingly recognized. It has become apparent that direct secretion of cholesterol from the blood compartment into the intestine, or transintestinal cholesterol excretion, plays a major role in disposal of cholesterol via the feces. This review will discuss current knowledge on the physiology of cholesterol homeostasis, with emphasis on cholesterol absorption, cholesterol synthesis and fecal excretion, and therapeutic options for hypercholesterolemia. PMID:24461630

  4. [Basic mechanisms: absorption and excretion of cholesterol and other sterols].

    PubMed

    Cofan Pujol, Montserrat

    2014-01-01

    Cholesterol is of vital importance for vertebrate cell membrane structure and function. It is obvious that adequate regulation of cholesterol homeostasis is essential. Hypercholesterolemia promotes atherosclerosis and thereby represents a major risk factor for cardiovascular disease. The liver has been considered the major site of control in maintenance of cholesterol homeostasis. The liver facilitates clearance of (very) low density lipoprotein particles and cholesterol-containing chylomicron remnants, synthesizes cholesterol, synthesizes and secretes (nascent) high density lipoprotein particles, secretes cholesterol and bile salts to bile, and is involved in reverse cholesterol transport. In recent years, however, the importance of the intestine in many aspects of cholesterol physiology is increasingly recognized. It has become apparent that direct secretion of cholesterol from the blood compartment into the intestine, or transintestinal cholesterol excretion, plays a major role in disposal of cholesterol via the feces. This review will discuss current knowledge on the physiology of cholesterol homeostasis, with emphasis on cholesterol absorption, cholesterol synthesis and fecal excretion, and therapeutic options for hypercholesterolemia.

  5. All about Cholesterol

    MedlinePlus

    ... are several kinds of fats in your blood. • LDL cholesterol is sometimes called “bad” cholesterol. It can narrow ... medicine to manage blood fats. They help lower LDL cholesterol. They also help lower your risk for a ...

  6. Enzyme modification of platinum microelectrodes for detection of cholesterol in vesicle lipid bilayer membranes.

    PubMed

    Devadoss, Anando; Palencsár, M Simona; Jiang, Dechen; Honkonen, Michael L; Burgess, James D

    2005-11-15

    Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content. PMID:16285691

  7. Cholesterol-lowering effect of plant sterols.

    PubMed

    AbuMweis, Suhad S; Jones, Peter J H

    2008-12-01

    Plant sterols are plant components that have a chemical structure similar to cholesterol except for the addition of an extra methyl or ethyl group; however, plant sterol absorption in humans is considerably less than that of cholesterol. In fact, plant sterols reduce cholesterol absorption and thus reduce circulating levels of cholesterol. Earlier studies that have tested the efficacy of plant sterols as cholesterol-lowering agents incorporated plant sterols into fat spreads. Later on, plant sterols were added to other food matrices, including juices, nonfat beverages, milk and yogurt, cheese, meat, croissants and muffins, and cereal and chocolate bars. The beneficial physiologic effects of plant sterols could be further enhanced by combining them with other beneficial substances, such as olive and fish oils, fibers, and soy proteins, or with exercise. The addition of plant sterols to the diet is suggested by health experts as a safe and effective way to reduce the risk of coronary heart disease. PMID:18937893

  8. Cholesterol homeostasis in cardiovascular disease and recent advances in measuring cholesterol signatures.

    PubMed

    Seo, Hong Seog; Choi, Man Ho

    2015-09-01

    Despite the biochemical importance of cholesterol, its abnormal metabolism has serious cellular consequences that lead to endocrine disorders such as cardiovascular disease (CVD). Nevertheless, the impact of blood cholesterol as a CVD risk factor is still debated, and treatment with cholesterol-lowering drugs remains controversial, particularly in older patients. Although, the prevalence of CVD increases with age, the underlying mechanisms for this phenomenon are not well understood, and metabolic changes have not been confirmed as predisposing factors of atherogenesis. The quantification of circulating biomarkers for cholesterol homeostasis is therefore warranted, and reference values for cholesterol absorption and synthesis should be determined in order to establish CVD risk factors. The traditional lipid profile is often derived rather than directly measured and lacks a universal standard to interpret the results. In contrast, mass spectrometry-based cholesterol profiling can accurately measure free cholesterol as a biologically active component. This approach allows to detect alterations in various metabolic pathways that control cholesterol homeostasis, by quantitative analysis of cholesterol and its precursors/metabolites as well as dietary sterols. An overview of the mechanism of cholesterol homeostasis under different physiological conditions may help to identify predictive biomarkers of concomitant atherosclerosis and conventional CVD risk factors.

  9. A DNA-Assembled Fe3O4@Ag Nanorod in Silica Matrix for Cholesterol Biosensing

    NASA Astrophysics Data System (ADS)

    Satvekar, R. K.; Tiwari, A. P.; Rohiwal, S. S.; Tiwale, B. M.; Pawar, S. H.

    2015-12-01

    A novel nanocomposite having DNA-assembled Fe3O4@Ag nanorods in silica matrix has been proposed for fabrication of bienzymatic cholesterol nanobiosensor. Cholesterol oxidase and horseradish peroxidase have been co-encapsulated in Silica/Fe3O4@Ag-DNA nanocomposite deposited on the indium tin oxide electrode. Cyclic voltammetry was employed for the electrochemical behavior of proposed biosensor and used to estimate cholesterol with a linear range of 5-195 mg/dL.

  10. Cholesterol testing and results

    MedlinePlus

    ... VLDL cholesterol) Lipoproteins are made of fat and protein. They carry cholesterol, triglycerides, and other fats, called ... Pencina MJ, Navar-Boggan AM, D'Agostino RB Sr, Williams K, Neely B, Sniderman AD, Peterson ED. ...

  11. Cholesterol and Plants

    ERIC Educational Resources Information Center

    Behrman, E. J.; Gopalan, Venkat

    2005-01-01

    There is a widespread belief among the public and even among chemist that plants do not contain cholesterol. This wrong belief is the result of the fact that plants generally contain only small quantities of cholesterol and that analytical methods for the detection of cholesterol in this range were not developed until recently.

  12. Antibodies to cholesterol.

    PubMed Central

    Swartz, G M; Gentry, M K; Amende, L M; Blanchette-Mackie, E J; Alving, C R

    1988-01-01

    Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained. Images PMID:3162316

  13. Avoiding Christmas cholesterol.

    PubMed

    1991-12-01

    Judging from your response to our September feature on cholesterol testing providing dietary advice has become of paramount importance to OHNs. The Flora Project for Heart Disease Prevention offers information on the risk factors of high cholesterol and has become a major noninstitutional authority on coronary heart disease. With Yuletide in sight The Flora Project offers advice on a cholesterol-clear Christmas.

  14. Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

    PubMed

    Pörn, M I; Slotte, J P

    1990-10-01

    Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin.

  15. Degradation of plasma membrane phosphatidylcholine appears not to affect the cellular cholesterol distribution.

    PubMed

    Pörn, M I; Ares, M P; Slotte, J P

    1993-08-01

    To clarify the role of possible cholesterol/phosphatidylcholine interactions in cellular cholesterol distribution, we have used a phosphatidylcholine-specific phospholipase C from Bacillus cereus to degrade the cell surface phosphatidylcholine of cultured human fibroblasts. Of cellular phosphatidylcholine, approximately 15% was susceptible to degradation by the phospholipase. In spite of the dramatic redistribution of cellular cholesterol that can be observed after sphingomyelin depletion, the degradation of cell surface phosphatidylcholine did not affect the distribution of cholesterol in fibroblasts. In cholesterol-depleted cells as well as in cholesterol-loaded cells, the size of the cell surface cholesterol pool (susceptible to cholesterol oxidase) remained unchanged after phosphatidylcholine degradation. The rate of cholesterol esterification with [3H]oleic acid and the rate of [3H]cholesterol efflux from fibroblasts to high density lipoproteins also remained unchanged after degradation of plasma membrane phosphatidylcholine. An increase in the level of [3H]cholesterol efflux to high density lipoproteins was observed after degradation of plasma membrane sphingomyelin with exogenous sphingomyelinase, in-contrast to earlier reports, where no such effect was observed. The results suggest that interactions between cholesterol and phosphatidylcholine in the fibroblast plasma membranes are less important than cholesterol/sphingomyelin interactions for the asymmetric distribution of cellular cholesterol.

  16. A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2014-01-01

    The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.

  17. Effect of plant sterol-enriched diets on plasma and egg yolk cholesterol concentrations and cholesterol metabolism in laying hens.

    PubMed

    Liu, X; Zhao, H L; Thiessen, S; House, J D; Jones, P J H

    2010-02-01

    Egg exists as a major dietary source of cholesterol in Western diets. In North America, laying hen diets are usually devoid of cholesterol when diets are formulated to exclude animal-based products. Hence, laying hens meet their physiological cholesterol requirement through de novo synthesis. Plant sterols exert a cholesterol-lowering effect in humans by interfering with intestinal sterol absorption. However, it is unknown whether plant sterol supplementation could be effective in reducing intestinal reabsorption of biliary cholesterol in laying hens, thus modulating whole body cholesterol in favor of lower plasma and yolk cholesterol content. The current study was designed to investigate the effect of diets enriched with 0, 0.5, 1, and 2% plant sterols on cholesterol absorption, synthesis, as well as plasma, liver, and egg yolk cholesterol concentrations in laying hens. After 8 wk of plant sterol intervention (first 2 wk were acclimatization), feed intake, BW, egg weight, egg yolk weight, egg production, Haugh units, liver mass, plasma, and hepatic cholesterol concentrations did not differ as a function of plant sterol supplementation. Egg cholesterol concentrations (mg/g) fluctuated during the 6-wk experimental period. At wk 6, a minor reduction in egg yolk cholesterol concentration (mg per g of yolk, P<0.05, vs. control) was observed in hens fed 1 and 2% cholesterol-enriched diets, respectively. However, such result failed to affect total egg cholesterol content. No statistical difference was observed across treatments over 6 wk. Neither cholesterol absorption rates nor synthesis differed as a function of treatment. Results suggested that overall cholesterol content in egg yolk was not affected by feeding hens plant sterol-enriched diets over 6 wk. PMID:20075279

  18. Chemically fashioned ZnO nanowalls and their potential application for potentiometric cholesterol biosensor

    NASA Astrophysics Data System (ADS)

    Israr, M. Q.; Sadaf, J. R.; Nur, O.; Willander, M.; Salman, S.; Danielsson, B.

    2011-06-01

    Chemically fashioned zinc oxide (ZnO) nanowalls on aluminum wire have been characterized and utilized to fabricate a potentiometric cholesterol biosensor by an electrostatic conjugation with cholesterol oxidase. The sensitivity, specificity, reusability, and stability of the conjugated surface of ZnO nanowalls with thickness of ˜80 nm have been investigated over a wide logarithmic concentrations of cholesterol electrolyte solution ranging from 1×10-6-1×10-3 M. The presented biosensor illustrates good linear sensitivity slope curve (˜53 mV/decade) corresponding to cholesterol concentrations along with rapid output response time of ˜5 s.

  19. Cholesterol in the retina: the best is yet to come

    PubMed Central

    Pikuleva, Irina A.; Curcio, Christine A.

    2014-01-01

    Historically understudied, cholesterol in the retina is receiving more attention now because of genetic studies showing that several cholesterol-related genes are risk factors for age-related macular degeneration (AMD) and because eye pathology studies showing high cholesterol content of drusen, aging Bruch's membrane, and newly found subretinal lesions. The challenge before us is determining how the cholesterol-AMD link is realized. Meeting this challenge will require an excellent understanding these genes’ roles in retinal physiology and how chorioretinal cholesterol is maintained. In the first half of this review, we will succinctly summarize physico-chemical properties of cholesterol, its distribution in the human body, general principles of maintenance and metabolism, and differences in cholesterol handling in human and mouse that impact on experimental approaches. This information will provide a backdrop to the second part of the review focusing on unique aspects of chorioretinal cholesterol homeostasis, aging in Bruch's membrane, cholesterol in AMD lesions, a model for lesion biogenesis, a model for macular vulnerability based on vascular biology, and alignment of AMD-related genes and pathobiology using cholesterol and an atherosclerosis-like progression as unifying features. We conclude with recommendations for the most important research steps we can take towards delineating the cholesterol-AMD link. PMID:24704580

  20. Home-Use Tests - Cholesterol

    MedlinePlus

    ... this test does: This is a home-use test kit to measure total cholesterol. What cholesterol is: Cholesterol is a fat (lipid) in your blood. High-density lipoprotein (HDL) ("good" cholesterol) helps protect your heart, but low-density lipoprotein (LDL) ("bad" cholesterol) can clog the arteries of your ...

  1. Children and Cholesterol

    MedlinePlus

    ... a coronary artery procedure; or who suffered a heart attack or sudden cardiac death before age 55. Those with a parent who has a history of high total cholesterol levels (240 mg/dL or higher). Talk to your child’s pediatrician ... Risk Calculator Printable Cholesterol Information Sheets Heart360 Health ...

  2. Cholesterol and Affective Morbidity

    PubMed Central

    Fiedorowicz, Jess G.; Palagummi, Narasimha M.; Behrendtsen, Ole; Coryell, William H.

    2009-01-01

    Depression and mania have been linked with low cholesterol though there has been limited prospective study of cholesterol and subsequent course of affective illness. We studied the relationship between fasting total cholesterol and subsequent depressive and manic symptoms. A total of 131 participants from a prospective cohort study were identified as having had a fasting total cholesterol evaluation at intake. Participants were predominantly inpatients at index visit and were followed for a median of 20 and up to 25 years. Cholesterol was modeled with age, gender, and index use of a mood stabilizer in linear regression to assess its influence on subsequent depressive symptom burden in participants with unipolar disorder as well as depressive and manic symptom burden in participants with bipolar disorder. Among bipolar participants (N=65), low cholesterol predicted a higher proportion of follow-up weeks with manic, but not depressive symptoms. Cholesterol did not appear to predict depressive symptom burden among participants with unipolar depression (N=66). Lower cholesterol levels may predispose individuals with bipolar disorder to a greater burden of manic symptomatology and may provide some insight into the underlying neurobiology. PMID:19969372

  3. Cholesterol and Your Child

    MedlinePlus

    ... traveling together are called lipoproteins . Two kinds — low-density lipoprotein (LDL) and high-density lipoprotein (HDL) — are the ones that most of us have heard about. Low-density lipoproteins , or "bad cholesterol," are the primary cholesterol ...

  4. Electrochemical oxidation of cholesterol

    PubMed Central

    2015-01-01

    Summary Indirect cholesterol electrochemical oxidation in the presence of various mediators leads to electrophilic addition to the double bond, oxidation at the allylic position, oxidation of the hydroxy group, or functionalization of the side chain. Recent studies have proven that direct electrochemical oxidation of cholesterol is also possible and affords different products depending on the reaction conditions. PMID:25977713

  5. What Your Cholesterol Levels Mean

    MedlinePlus

    ... Pressure Tools & Resources Stroke More What Your Cholesterol Levels Mean Updated:Aug 17,2016 How’s your cholesterol? Time to get it checked! Keeping your cholesterol levels healthy is a great way to keep your ...

  6. CHARACTERISTICS OF POLYPHENOL OXIDASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones. Highly reactive o-quinones couple with phenolics and specific amino acids on proteins to form the characteristic browning products in many wounded fruits, vegetables, and leaf tissues of plant...

  7. Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase

    PubMed Central

    Le Laz, Sébastien; kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2016-01-01

    Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed. PMID:26815910

  8. Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase.

    PubMed

    Le Laz, Sébastien; Kpebe, Arlette; Bauzan, Marielle; Lignon, Sabrina; Rousset, Marc; Brugna, Myriam

    2016-01-01

    Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed. PMID:26815910

  9. Cholesterol and Ion Channels

    PubMed Central

    Levitan, Irena; Fang, Yun; Rosenhouse-Dantsker, Avia; Romanenko, Victor

    2010-01-01

    A variety of ion channels, including members of all major ion channel families, have been shown to be regulated by changes in the level of membrane cholesterol and partition into cholesterol-rich membrane domains. In general, several types of cholesterol effects have been described. The most common effect is suppression of channel activity by an increase in membrane cholesterol, an effect that was described for several types of inwardly-rectifying K+ channels, voltage-gated K+ channels, Ca+2 sensitive K+ channels, voltage-gated Na+ channels, N-type voltage-gated Ca+2 channels and volume-regulated anion channels. In contrast, several types of ion channels, such as epithelial amiloride-sensitive Na+ channels and Transient Receptor Potential channels, as well as some of the types of inwardly-rectifying and voltage-gated K+ channels were shown to be inhibited by cholesterol depletion. Cholesterol was also shown to alter the kinetic properties and current-voltage dependence of several voltage-gated channels. Finally, maintaining membrane cholesterol level is required for coupling ion channels to signalling cascades. In terms of the mechanisms, three general mechanisms have been proposed: (i) specific interactions between cholesterol and the channel protein, (ii) changes in the physical properties of the membrane bilayer and (iii) maintaining the scaffolds for protein-protein interactions. The goal of this review is to describe systematically the role of cholesterol in regulation of the major types of ion channels and to discuss these effects in the context of the models proposed. PMID:20213557

  10. NADPH Oxidases in Lung Health and Disease

    PubMed Central

    Bernard, Karen; Hecker, Louise; Luckhardt, Tracy R.; Cheng, Guangjie

    2014-01-01

    Abstract Significance: The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. Recent advances: The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. Critical issues: However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. Future Directions: Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease. Antioxid. Redox Signal. 20, 2838–2853. PMID:24093231

  11. The mechanism of cytochrome C oxidase inhibition by nitric oxide.

    PubMed

    Antunes, Fernando; Cadenas, Enrique

    2007-01-01

    The basic biochemistry of the inhibition of cytochrome oxidase by NO is reviewed. Three possible mechanisms that include the binding of NO to the fully reduced Fe(a3)-Cu(B) site, to the semi-reduced Fe(a3)-Cu(B) site, and to the fully oxidized Fe(a3)-Cu(B) site are confronted with the experimental data. Mathematical models are used to facilitate the analysis and to solve puzzling observations concerning the NO inhibition of cytochrome oxidase. It is concluded that the inhibition of cytochrome oxidase by NO is mixed, having both competitive and uncompetitive components, but under physiological electron flows the competitive component is largely predominant. The physiological and pathological relevance of this inhibition is briefly discussed.

  12. Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.

    PubMed

    Maekawa, Masashi; Fairn, Gregory D

    2015-04-01

    Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles. PMID:25663704

  13. Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol.

    PubMed

    Maekawa, Masashi; Fairn, Gregory D

    2015-04-01

    Cholesterol is an essential component of metazoan cellular membranes and it helps to maintain the structural integrity and fluidity of the plasma membrane. Here, we developed a cholesterol biosensor, termed D4H, based on the fourth domain of Clostridium perfringens theta-toxin, which recognizes cholesterol in the cytosolic leaflet of the plasma membrane and organelles. The D4H probe disassociates from the plasma membrane upon cholesterol extraction and after perturbations in cellular cholesterol trafficking. When used in combination with a recombinant version of the biosensor, we show that plasmalemmal phosphatidylserine is essential for retaining cholesterol in the cytosolic leaflet of the plasma membrane. In vitro experiments reveal that 1-stearoy-2-oleoyl phosphatidylserine can induce phase separation in cholesterol-containing lipid bilayers and shield cholesterol from cholesterol oxidase. Finally, the altered transbilayer distribution of cholesterol causes flotillin-1 to relocalize to endocytic organelles. This probe should be useful in the future to study pools of cholesterol in the cytosolic leaflet of the plasma membrane and organelles.

  14. Dietary Fat and Cholesterol

    MedlinePlus

    ... Gynecology Medical Conditions Nutrition & Fitness Emotional Health Dietary Fat and Cholesterol Posted under Health Guides . Updated 23 ... warm What are the different types of dietary fat? The four main types of fat found in ...

  15. Get Your Cholesterol Checked

    MedlinePlus

    ... is checked with a blood test called a lipid profile. During the test, a nurse will take ... blood tests that can check cholesterol, but a lipid profile gives the most information. Find out more ...

  16. High Blood Cholesterol

    MedlinePlus

    ... of cholesterol is called plaque. Plaque Buildup Can Lead to… Click for more information Artherosclerosis. Over time, ... disease (CHD). Angina. The buildup of plaque can lead to chest pain called angina. Angina is a ...

  17. Common Misconceptions about Cholesterol

    MedlinePlus

    ... most (and preferably all) days; and stressing the importance of avoiding tobacco products. Learn more about cholesterol ... Privacy Policy Popular Articles 1 Understanding Blood Pressure Readings 2 Sodium and Salt 3 Low Blood Pressure ...

  18. Cholesterol and Statins

    MedlinePlus

    ... the liver makes ldl & hdl In the liver, triglycerides, cholesterol, and proteins form together to make LDL ... This is especially important for individuals with high triglyceride and/or low HDL levels who are overweight ...

  19. Compounds affecting cholesterol absorption

    NASA Technical Reports Server (NTRS)

    Hua, Duy H. (Inventor); Koo, Sung I. (Inventor); Noh, Sang K. (Inventor)

    2004-01-01

    A class of novel compounds is described for use in affecting lymphatic absorption of cholesterol. Compounds of particular interest are defined by Formula I: ##STR1## or a pharmaceutically acceptable salt thereof.

  20. The Success Story of LDL Cholesterol Lowering.

    PubMed

    Pedersen, Terje R

    2016-02-19

    We can look back at >100 years of cholesterol research that has brought medicine to a stage where people at risk of severe or fatal coronary heart disease have a much better prognosis than before. This progress has not come about without resistance. Perhaps one of the most debated topics in medicine, the cholesterol controversy, could only be brought to rest through the development of new clinical research methods that were capable of taking advantage of the amazing achievements in basic and pharmacological science after the second World War. It was only after understanding the biochemistry and physiology of cholesterol synthesis, transport and clearance from the blood that medicine could take advantage of drugs and diets to reduce the risk of atherosclerotic diseases. This review points to the highlights of the history of low-density lipoprotein-cholesterol lowering, with the discovery of the low-density lipoprotein receptor and its physiology and not only the development of statins as the stellar moments but also the development of clinical trial methodology as an effective tool to provide scientifically convincing evidence. PMID:26892969

  1. MD-2 binds cholesterol.

    PubMed

    Choi, Soo-Ho; Kim, Jungsu; Gonen, Ayelet; Viriyakosol, Suganya; Miller, Yury I

    2016-02-19

    Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4). Here we demonstrate that cholesterol binds to myeloid differentiation-2 (MD-2), a TLR4 ancillary molecule, which is a binding receptor for bacterial lipopolysaccharide (LPS) and is indispensable for LPS-induced TLR4 dimerization and signaling. Cholesterol binding to MD-2 was competed by LPS and by OxCE-modified BSA. Furthermore, soluble MD-2 in human plasma and MD-2 in mouse atherosclerotic lesions carried cholesterol, the finding supporting the biological significance of MD-2 cholesterol binding. These results help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis.

  2. Enzymatic assay of total cholesterol in serum or plasma by amperometric measurement of rate of oxygen depletion following saponification.

    PubMed

    Kumar, A; Christian, G D

    1977-01-17

    A method for serum or plasma cholesterol assay involving amperometric measurement of the rate of oxygen depletion in the cholesterol oxidase-catalyzed oxidation of cholesterol is described. The hydrolysis of the serum cholesterol esters is accomplished by saponification of 50 mul of sample with 0.2 ml of ethanolic KOH (1.0 mol/1) containing 0.5% Triton X-100 for 5 min at 75 degrees C. The rate of oxygen consumption in a 25-mul aliquot of this is measured with a Clark electrode in a Beckman Glucose Analyzer and the assay takes about one minute after incubation; results are read digitally on the instrument. The analyzer cell contains 1 ml of 1 M phosphate buffer, pH 7.4, with 100 mg sodium cholate/100 ml and 0.1-0.2 U cholesterol oxidase.

  3. Alternative oxidase and uncoupling protein: thermogenesis versus cell energy balance.

    PubMed

    Jarmuszkiewicz, W; Sluse-Goffart, C M; Vercesi, A E; Sluse, F E

    2001-04-01

    The physiological role of an alternative oxidase and an uncoupling protein in plant and protists is discussed in terms of thermogenesis and energy metabolism balance in the cell. It is concluded that thermogenesis is restricted not only by a lower-limit size but also by a kinetically-limited stimulation of the mitochondrial respiratory chain.

  4. Europium tetracycline biosensor for the determination of cholesterol

    NASA Astrophysics Data System (ADS)

    Courrol, Lilia Coronato; Silva, Flávia Rodrigues de Oliveira; Samad, Ricardo Elgul; Mansano, Ronaldo Domingues; Vieira, Nilson Dias, Jr.

    2007-02-01

    Development of cholesterol biosensors is of great importance in clinical analysis because the concentration of cholesterol in blood is a fundamental parameter for the prevention and diagnosis of a number of clinical disorders such as heart disease, hypertension and arteriosclerosis. In general, determination of cholesterol is based on spectrophotometry; but this method involves complicated procedures and the cost is high because expensive enzyme must be used in each assay. We report here the observation, for the first time, of the enhancement of Europium-Tetracycline complex emission in cholesterol solutions. This enhancement was initially observed with the addition of the enzyme cholesterol oxidase, which produces H IIO II, the agent driver of the Europium tetracycline complex, to the solution. However, it was found that the enzyme is not needed to enhance the luminescence. A calibration curve was determined, resulting in an easy-handling immobilization method with a cheap stable material. This method shows that the complex can be used as a sensor to determine cholesterol in biological systems with good selectivity, fast response, miniature size, and reproducible results.

  5. Cholesterol Absorption and Metabolism.

    PubMed

    Howles, Philip N

    2016-01-01

    Inhibitors of cholesterol absorption have been sought for decades as a means to treat and prevent cardiovascular diseases (CVDs) associated with hypercholesterolemia. Ezetimibe is the one clear success story in this regard, and other compounds with similar efficacy continue to be sought. In the last decade, the laboratory mouse, with all its genetic power, has become the premier experimental model for discovering the mechanisms underlying cholesterol absorption and has become a critical tool for preclinical testing of potential pharmaceutical entities. This chapter briefly reviews the history of cholesterol absorption research and the various gene candidates that have come under consideration as drug targets. The most common and versatile method of measuring cholesterol absorption is described in detail along with important considerations when interpreting results, and an alternative method is also presented. In recent years, reverse cholesterol transport (RCT) has become an area of intense new interest for drug discovery since this process is now considered another key to reducing CVD risk. The ultimate measure of RCT is sterol excretion and a detailed description is given for measuring neutral and acidic fecal sterols and interpreting the results. PMID:27150091

  6. An enzyme thermistor-based assay for total and free cholesterol.

    PubMed

    Raghavan, V; Ramanathan, K; Sundaram, P V; Danielsson, B

    1999-11-01

    A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit.

  7. Transfer of cholesterol from macrophages to lymphocytes in culture.

    PubMed

    de Bittencourt Júnior, P I; Curi, R

    1998-02-01

    -cultivation with macrophages decreased the basal incorporation of [2-14C]thymidine into lymphocyte DNA and the addition of cholesterol to lymphocyte culture media also impaired the lymphocyte proliferative response to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS). The above results suggest that macrophages may transfer cholesterol to lymphocytes (from both lymph nodes and blood), thus regulating lymphocyte function by raising the intracellular cholesterol content and suppressing lymphocyte proliferative activity. If this is so, a modulatory role for the transfer of cholesterol in both physiological (e.g. immune response) and pathological conditions (e.g. atherosclerosis) may be postulated. This hypothesis is currently under investigation in our laboratory.

  8. Cholesterol-Lowering Effect of Calcium Alginate in Rats.

    PubMed

    Idota, Yoko; Kogure, Yumi; Kato, Takako; Ogawa, Mana; Kobayashi, Shoko; Kakinuma, Chihaya; Yano, Kentaro; Arakawa, Hiroshi; Miyajima, Chihiro; Kasahara, Fumiyoshi; Ogihara, Takuo

    2016-01-01

    We examined whether calcium alginate (Ca-Alg) reduces blood cholesterol levels in rats fed a high-cholesterol diet. First, we examined taurocholate adsorption in vitro by various types of sodium alginate (Na-Alg). High molecular-weight, guluronic acid-rich Na-Alg showed the greatest adsorption of taurocholate, and therefore the corresponding Ca-Alg was chosen for the in vivo study. Rats were fed a high-cholesterol diet or a Ca-Alg-containing diet for 2 weeks. Body weight and diet intake were measured, and the general condition of the animals was monitored during this period. After 14 d, the plasma concentration of cholesterol, portal plasma concentration of bile acid, and bile acid in feces were measured. The plasma concentration of cholesterol was significantly reduced in rats fed a 2% Ca-Alg-containing diet. Furthermore, the portal concentration of bile acid was significantly lowered in the 2% Ca-Alg group. A tendency for a Ca-Alg concentration-dependent increase in fecal excretion of bile acid was also seen, although it was not statistically significant. While several changes in biochemical parameters and histopathological findings were observed, all the values remained within the physiological range. These results indicate that Ca-Alg is effective in reducing plasma cholesterol. A possible mechanism would be enhanced fecal excretion of bile acid due to reduced intestinal reabsorption, which in turn might stimulate bile acid synthesis from cholesterol in the liver, leading to a decrease in plasma cholesterol.

  9. Whole body and tissue cholesterol turnover in the baboon

    SciTech Connect

    Dell, R.B.; Mott, G.E.; Jackson, E.M.; Ramakrishnan, R.; Carey, K.D.; McGill, H.C. Jr.; Goodman, D.S.

    1985-03-01

    Cholesterol turnover was studied in four baboons by injecting (/sup 14/C)cholesterol 186 days and (/sup 3/H)cholesterol 4 days before necropsy, and fitting a two- or three-pool model to the resulting specific activity-time data. At necropsy, cholesterol mass and specific activity were determined for the total body and for many tissues. The principal aim of this study was to estimate the extent of cholesterol synthesis in the side pools of the model, by computing the amount of side pool synthesis needed to equal the measured total body cholesterol. Central pool synthesis varied from 61 to 89% of the total cholesterol production rate. Moreover, the finding that the measured total body cholesterol fell within the range obtained from the kinetic analysis by using reasonable assumptions, provides evidence for the physiological validity of the model. A second aim of this study was to explore cholesterol turnover in various tissues. A pool model predicts that rapidly turning over tissues will have higher specific activities at early times and lower specific activities at later times after injection of tracer relative to slowly turning over tissues, except where significant synthesis occurs. Results in all four baboons were similar. Turnover rates for the different tissues loosely fell into three groups which were turning over at fast, intermediate, and slow rates. Finally, the magnitude of variation of cholesterol specific activity was moderate for several distributed tissues (fat, muscle, arteries, and the alimentary tract), but was small for liver. Cholesterol turnover in serial biopsies of skin, muscle, and fat could, however, be fitted with a single pool to estimate tissue turnover rates.

  10. Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies

    PubMed Central

    Andjelković, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T.

    2015-01-01

    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. PMID:26672986

  11. CHOLESTEROL AND CHOLESTEROL ESTER CONTENT OF BOVINE COLOSTRUM

    PubMed Central

    Shope, Richard E.; Gowen, John W.

    1928-01-01

    The total amount of cholesterol found in colostrum and milk is comparatively low. The amount of cholesterol found in colostrum declines at an ever decreasing rate as milk secretion develops until at 48 hours the cholesterol is nearly the same as that found in milk 3 months or 7 months after parturition. The morning milk differs from the evening milk in that the cholesterol bound as ester is greater in amount. PMID:19869468

  12. Cholesterol: Up in Smoke.

    ERIC Educational Resources Information Center

    Raloff, Janet

    1991-01-01

    Discussed is the contribution cooked meat makes to air pollution. The dozens of compounds, including cholesterol, that are released when a hamburger is grilled are described. The potential effects of these emissions on humans and the urban environment are discussed. (KR)

  13. Cholesterol, inflammasomes, and atherogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasma cholesterol levels have been strongly associated with atherogenesis, underscoring the role of lipid metabolism in defining cardiovascular disease risk. However, atherosclerotic plaque is highly dynamic and contains elements of both the innate and adaptive immune system that respond to the abe...

  14. Niacin for cholesterol

    MedlinePlus

    ... this page, please enable JavaScript. Niacin is a B-vitamin. When taken as a prescription in larger doses, ... A.M. Editorial team. Related MedlinePlus Health Topics B Vitamins Cholesterol Browse the Encyclopedia A.D.A.M., ...

  15. Crystallization of Mitochondrial Cytochrome Oxidase

    NASA Astrophysics Data System (ADS)

    Ozawa, Takayuki; Tanaka, Masashi; Wakabayashi, Takashi

    1982-12-01

    Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 μ m in average length and 5 μ m in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 angstrom, b = 84 angstrom, and c = 103 angstrom, and α =β 90 degrees, γ = 126 degrees.

  16. Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives.

    PubMed

    Lecompte, Marie-France; Gaibelet, Gérald; Lebrun, Chantal; Tercé, François; Collet, Xavier; Orlowski, Stéphane

    2015-11-01

    Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus

  17. X-ray absorption spectroscopy of chicken sulfite oxidase crystals

    SciTech Connect

    George, G.N.; Pickering, I.J.; Kisker, C.

    1999-05-17

    Sulfite oxidase catalyzes the physiologically vital oxidation of sulfite to sulfate. Recently, the crystal structure of chicken sulfite oxidase has been reported at 1.9 {angstrom} resolution. In contrast to the information available from previous X-ray absorption spectroscopic studies, the active site indicated by crystallography was a mono-oxo species. Because of this the possibility that the crystals did in fact contain a reduced molybdenum species was considered in the crystallographic work. The authors report herein an X-ray absorption spectroscopic study of polycrystalline sulfite oxidase prepared in the same manner as the previous single-crystal samples, and compare this with data for frozen solutions of oxidized and reduced enzyme.

  18. Oxidation of polymines by diamine oxidase from human seminal plasma.

    PubMed Central

    Hölttä, E; Pulkkinen, P; Elfving, K; Jänne, J

    1975-01-01

    1. Diamine oxidase [amine-oxygen oxidoreductase (deaminating)(pyridoxal-containing), EC 1.4.3.6] was purified from human seminal plasma more than 1,700-fold. The enzyme appeared to be homogeneous on polyacrylamide-gel electrophoresis at two different pH values. 2. The general properties of the enzyme were comparable with those described for other diamine oxidases from different mammalian sources. The molecular weight of the enzyme was calculated to be about 182,000. 3. The enzyme had highest affinity for diamines, but polyamines spermidine and spermine were also degraded at concentrations that can be considered physiological in human semen. 3. The possible degradation of spermine by diamine oxidase in human semen in vivo may give rise to the formation of cytotoxic aldehydes that conceivably can influence the motility and survival of the spermatozoa. PMID:239684

  19. Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2015-07-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.

  20. Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

    PubMed Central

    Yue, Hai-Yuan; Xu, Jianhua

    2015-01-01

    Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

  1. Unsubstituted phenothiazine as a superior water-insoluble mediator for oxidases.

    PubMed

    Sekretaryova, Alina N; Vagin, Mikhail Yu; Beni, Valerio; Turner, Anthony P F; Karyakin, Arkady A

    2014-03-15

    The mediation of oxidases glucose oxidase (GOx), lactate oxidase (LOx) and cholesterol oxidase (ChOx) by a new electron shuttling mediator, unsubstituted phenothiazine (PTZ), was studied. Cyclic voltammetry and rotating-disk electrode measurements in nonaqueous media were used to determine the diffusion characteristics of the mediator and the kinetics of its reaction with GOx, giving a second-order rate constant of 7.6 × 10(3)-2.1 × 10(4)M(-1)s(-1) for water-acetonitrile solutions containing 5-15% water. These values are in the range reported for commonly used azine-type mediators, indicating that PTZ is able to function as an efficient mediator. PTZ and GOx, LOx and ChOx were successfully co-immobilised in sol-gel membrane on a screen-printed electrode to construct glucose, lactate and cholesterol biosensors, respectively, which were then optimised in terms of stability and sensitivity. The electrocatalytic oxidation responses showed a dependence on substrate concentration ranging from 0.6 to 32 mM for glucose, from 19 to 565 mM for lactate and from 0.015 to 1.0mM for cholesterol detection. Oxidation of substrates on the surface of electrodes modified with PTZ and enzyme membrane was investigated with double-step chronoamperometry and the results showed that the PTZ displays excellent electrochemical catalytic activities even when immobilised on the surface of the electrode.

  2. Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

    PubMed

    Nomura, Johji; Busso, Nathalie; Ives, Annette; Matsui, Chieko; Tsujimoto, Syunsuke; Shirakura, Takashi; Tamura, Mizuho; Kobayashi, Tsunefumi; So, Alexander; Yamanaka, Yoshihiro

    2014-04-01

    Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

  3. Understand Your Risk for High Cholesterol

    MedlinePlus

    ... or trans fats also increases the amount of LDL cholesterol in your blood. If high blood cholesterol runs ... may not be enough to help lower your LDL blood cholesterol. View an animation of cholesterol . More information: Women ...

  4. Overview of Cholesterol and Lipid Disorders

    MedlinePlus

    ... Cholesterol and Lipid Disorders Dyslipidemia Hypolipidemia Cholesterol and triglycerides are important fats (lipids) in the blood. Cholesterol ... needs, but it also obtains cholesterol from food. Triglycerides, which are contained in fat cells, can be ...

  5. Sulfide inhibition of and metabolism by cytochrome c oxidase.

    PubMed

    Nicholls, Peter; Marshall, Doug C; Cooper, Chris E; Wilson, Mike T

    2013-10-01

    Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal ('gasotransmitter') species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable 'substrate'. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the 'pulsed' and 'resting' states. The pulsed form reacts aerobically with sulfide to form two intermediates, 'P' and 'F', otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a. In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented. PMID:24059525

  6. Amperometric cholesterol biosensors based on the electropolymerization of pyrrole and aniline in sulphuric Acid for the determination of cholesterol in serum.

    PubMed

    Muhammet, Sinan M; Cete, Servet; Arslan, Fatma; Yaşar, Ahmet

    2009-01-01

    A new amperometric cholesterol biosensor was prepared by immobilizing cholesterol oxidase by a glutaraldehyde crosslinking procedure on polypyrrole-polyaniline (ppy-pani) composite film on the surface of a platinum electrode. In order to prepare a biosensor for the determination of cholesterol, electropolymerization of pyrrole and aniline on Pt surface was performed with an electrochemical cell containing pyrrole and aniline in sulphuric acid by cyclic voltammetry between 0.0 and 0,7 V (vs.Ag/AgCl) at a scan rate of 50 mV upon Pt electrode. The amperometric determination is based on the electrochemical detection of H(2)O(2), which is generated in enzymatic reaction of cholesterol. The cholesterol determined by the oxidation of enzymatically generated H(2)O(2) at 0.7 V vs. Ag/AgCl. The optimized cholesterol oxidase biosensor displayed linear working range and a response time of 300 s. The effects of pH and temperature were investigated and optimum parameters were found to be 7.0, 25 degrees C, respectively. In addition to this, the stability and reproducibility of biosensor were tried. Operational stability of the proposed cholesterol biosensor was obtained by periodical measurements of the biosensor response. Biosensor at optimum activity conditions was used in 30 activity assays in one day to determine the operational stability. The results show that 82% of the response current was retained after 30 activity assays. The electrode was stored in a refrigerator at 4 degrees C after the measurements. The storage stability of the biosensor was determined by performing activity assays within 23 days. The results demonstrate that 60% of the response current was retained after 23 days. Preparing biosensor is used for the analysis of cholesterol in serum.

  7. Cholesterol Metabolism in CKD.

    PubMed

    Reiss, Allison B; Voloshyna, Iryna; De Leon, Joshua; Miyawaki, Nobuyuki; Mattana, Joseph

    2015-12-01

    Patients with chronic kidney disease (CKD) have a substantial risk of developing coronary artery disease. Traditional cardiovascular disease (CVD) risk factors such as hypertension and hyperlipidemia do not adequately explain the high prevalence of CVD in CKD. Both CVD and CKD are inflammatory states and inflammation adversely affects lipid balance. Dyslipidemia in CKD is characterized by elevated triglyceride levels and high-density lipoprotein levels that are both decreased and dysfunctional. This dysfunctional high-density lipoprotein becomes proinflammatory and loses its atheroprotective ability to promote cholesterol efflux from cells, including lipid-overloaded macrophages in the arterial wall. Elevated triglyceride levels result primarily from defective clearance. The weak association between low-density lipoprotein cholesterol level and coronary risk in CKD has led to controversy over the usefulness of statin therapy. This review examines disrupted cholesterol transport in CKD, presenting both clinical and preclinical evidence of the effect of the uremic environment on vascular lipid accumulation. Preventative and treatment strategies are explored. PMID:26337134

  8. Cholesterol binding to ion channels

    PubMed Central

    Levitan, Irena; Singh, Dev K.; Rosenhouse-Dantsker, Avia

    2014-01-01

    Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions. PMID:24616704

  9. Increased steroid hormone secretion in mouse Leydig tumor cells after induction of cholesterol translocation by sphingomyelin degradation.

    PubMed

    Pörn, M I; Tenhunen, J; Slotte, J P

    1991-06-01

    The effects of sphingomyelin degradation on [3H]cholesterol transfer from the cell surface to mitochondria were examined in mouse Leydig tumor cells. These cells were used since they utilize cholesterol for steroid hormone synthesis in the mitochondria, and also possess acyl-CoA: cholesterol acyl transferase (ACAT) activity in the endoplasmic reticulum. Exposure of glutaraldehyde-fixed mouse Leydig tumor cells to sphingomyelinase (50 mU/ml, 60 min) resulted in the degradation of about 50% of cell sphingomyelin, suggesting that only half of the sphingomyelin mass in these cells was located in the exoleaflet of the plasma membrane. The partial sphingomyelin degradation resulted in the translocation of cellular unesterified [3H]cholesterol from plasma membranes (cholesterol oxidase-susceptible) to intracellular compartments (oxidase-resistant). The fraction of [3H]cholesterol that was translocated, i.e., between 20 and 50%, varied with different [3H]cholesterol-labeling methods. Cholesterol translocation induced by sphingomyelin degradation subsequently led to the stimulation of ACAT activity, suggesting that a fraction of cell surface cholesterol was transported to the endoplasmic reticulum. The sphingomyelinase-induced [3H]cholesterol flow from the cell surface to the cell interior was also in part directed to the mitochondria, as evidenced by the increased secretion of [3H]steroid hormones. In addition, the cyclic AMP-induced activation of steroidogenesis was further enhanced by the sphingomyelinase-induced cholesterol translocation. Based on the current results, it seems evident that a significant portion of the translocated [3H]cholesterol made its way from plasma membranes into the mitochondria for steroidogenesis.

  10. Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2 155.

    PubMed

    Uhía, I; Galán, B; Morales, V; García, J L

    2011-04-01

    The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process. PMID:21208358

  11. Nox NADPH Oxidases and the Endoplasmic Reticulum

    PubMed Central

    Araujo, Thaís L.S.; Abrahão, Thalita B.

    2014-01-01

    Abstract Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between

  12. Indole-3-ethanol Oxidase

    PubMed Central

    Percival, Frank W.; Purves, William K.; Vickery, Larry E.

    1973-01-01

    We report the further characterization of indole-3-ethanol oxidase from cucumber seedlings. The effects of various inhibitors suggest that the enzyme may be a flavoprotein with a metal ion and sulfhydryl groups required for full activity. Indole-3-acetaldehyde, a product of the reaction, inhibits the enzyme. This inhibition is overcome by O2 but not by indole-3-ethanol, indicating that the kinetic mechanism of the enzyme is a ping-pong Bi-Bi. The enzyme undergoes cooperative interactions with indoleethanol, yielding Hill coefficients as high as 2.96. Gibberellins are without effect on the enzyme, but it is inhibited by several acidic indoles possessing growth-promoting activity and by two synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. Increasing concentrations of indoleacetic acid (IAA) brought about a slight reduction in the indoleethanol concentration producing halfmaximal velocity. Increasing levels of indoleethanol decreased the concentration of IAA required for half-maximal inhibition. At low concentrations of indoleethanol, low levels of IAA activated rather than inhibited. The effect of IAA was not overcome at higher levels of indoleethanol. These results may be interpreted as showing that IAA is a noncompetitive inhibitor which binds to that conformation of the enzyme which also binds indoleethanol. The significance of these interactions for the regulation of IAA biosynthesis is discussed. PMID:16658401

  13. Brain Cholesterol Metabolism and Its Defects: Linkage to Neurodegenerative Diseases and Synaptic Dysfunction

    PubMed Central

    Petrov, A. M.; Kasimov, M. R.; Zefirov, A. L.

    2016-01-01

    Cholesterol is an important constituent of cell membranes and plays a crucial role in the compartmentalization of the plasma membrane and signaling. Brain cholesterol accounts for a large proportion of the body’s total cholesterol, existing in two pools: the plasma membranes of neurons and glial cells and the myelin membranes . Cholesterol has been recently shown to be important for synaptic transmission, and a link between cholesterol metabolism defects and neurodegenerative disorders is now recognized. Many neurodegenerative diseases are characterized by impaired cholesterol turnover in the brain. However, at which stage the cholesterol biosynthetic pathway is perturbed and how this contributes to pathogenesis remains unknown. Cognitive deficits and neurodegeneration may be associated with impaired synaptic transduction. Defects in cholesterol biosynthesis can trigger dysfunction of synaptic transmission. In this review, an overview of cholesterol turnover under physiological and pathological conditions is presented (Huntington’s, Niemann-Pick type C diseases, Smith-Lemli-Opitz syndrome). We will discuss possible mechanisms by which cholesterol content in the plasma membrane influences synaptic processes. Changes in cholesterol metabolism in Alzheimer’s disease, Parkinson’s disease, and autistic disorders are beyond the scope of this review and will be summarized in our next paper. PMID:27099785

  14. Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase.

    PubMed

    McDonald, Allison E; Amirsadeghi, Sasan; Vanlerberghe, Greg C

    2003-12-01

    The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.

  15. Food combinations for cholesterol lowering.

    PubMed

    Harland, Janice I

    2012-12-01

    Reducing elevated LDL-cholesterol is a key public health challenge. There is substantial evidence from randomised controlled trials (RCT) that a number of foods and food components can significantly reduce LDL-cholesterol. Data from RCT have been reviewed to determine whether effects are additive when two or more of these components are consumed together. Typically components, such as plant stanols and sterols, soya protein, β-glucans and tree nuts, when consumed individually at their target rate, reduce LDL-cholesterol by 3-9 %. Improved dietary fat quality, achieved by replacing SFA with unsaturated fat, reduces LDL-cholesterol and can increase HDL-cholesterol, further improving blood lipid profile. It appears that the effect of combining these interventions is largely additive; however, compliance with multiple changes may reduce over time. Food combinations used in ten 'portfolio diet' studies have been reviewed. In clinical efficacy studies of about 1 month where all foods were provided, LDL-cholesterol is reduced by 22-30 %, whereas in community-based studies of >6 months' duration, where dietary advice is the basis of the intervention, reduction in LDL-cholesterol is about 15 %. Inclusion of MUFA into 'portfolio diets' increases HDL-cholesterol, in addition to LDL-cholesterol effects. Compliance with some of these dietary changes can be achieved more easily compared with others. By careful food component selection, appropriate to the individual, the effect of including only two components in the diet with good compliance could be a sustainable 10 % reduction in LDL-cholesterol; this is sufficient to make a substantial impact on cholesterol management and reduce the need for pharmaceutical intervention.

  16. Cholesterol in brain disease: sometimes determinant and frequently implicated

    PubMed Central

    Martín, Mauricio G; Pfrieger, Frank; Dotti, Carlos G

    2014-01-01

    Cholesterol is essential for neuronal physiology, both during development and in the adult life: as a major component of cell membranes and precursor of steroid hormones, it contributes to the regulation of ion permeability, cell shape, cell–cell interaction, and transmembrane signaling. Consistently, hereditary diseases with mutations in cholesterol-related genes result in impaired brain function during early life. In addition, defects in brain cholesterol metabolism may contribute to neurological syndromes, such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD), and even to the cognitive deficits typical of the old age. In these cases, brain cholesterol defects may be secondary to disease-causing elements and contribute to the functional deficits by altering synaptic functions. In the first part of this review, we will describe hereditary and non-hereditary causes of cholesterol dyshomeostasis and the relationship to brain diseases. In the second part, we will focus on the mechanisms by which perturbation of cholesterol metabolism can affect synaptic function. PMID:25223281

  17. Development of a platinized and ferrocene-mediated cholesterol amperometric biosensor based on electropolymerization of polypyrrole in a flow system.

    PubMed

    Vidal, Juan-C; Garcia, E; Castillo, Juan-R

    2002-05-01

    The preparation of a cholesterol amperometric biosensor using a platinized Pt electrode as a support for the electropolymerization of a polypyrrole film, in which cholesterol oxidase and ferrocene monocarboxylic acid (electron-transfer mediator) were co-entrapped, is described. All the biosensor preparation steps (platinization and electropolymerization) and the cholesterol determination take place in the same flow system. The presence of the mediator enhances the sensitivity and selectivity of the platinized biosensor without modifying the dynamic parameters of the response, and the platinized layer improves the operational lifetime of the mediated sensor. The sensitivity obtained was 88.51 nA mM(-1) and the limit of detection was 12.4 microM of cholesterol. The analytical properties of the biosensor for the flow-injection determination of cholesterol were studied and compared with those of other more simple amperometric biosensor configurations.

  18. How cholesterol regulates endothelial biomechanics

    PubMed Central

    Hong, Zhongkui; Staiculescu, Marius C.; Hampel, Paul; Levitan, Irena; Forgacs, Gabor

    2012-01-01

    As endothelial cells form the barrier between blood flow and surrounding tissue, many of their functions depend on mechanical integrity, in particular those of the plasma membrane. As component and organizer of the plasma membrane, cholesterol is a regulator of cellular mechanical properties. Disruption of cholesterol balance leads to impairment of endothelial functions and eventually to disease. The mechanical properties of the membrane are strongly affected by the cytoskeleton. As Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key mediator between the membrane and cytoskeleton, it also affects cellular biomechanical properties. Typically, PIP2 is concentrated in cholesterol-rich microdomains, such as caveolae and lipid rafts, which are particularly abundant in the endothelial plasma membrane. We investigated the connection between cholesterol and PIP2 by extracting membrane tethers from bovine aortic endothelial cells (BAEC) at different cholesterol levels and PIP2 conditions. Our results suggest that in BAEC the role of PIP2, as a mediator of membrane-cytoskeleton adhesion, is regulated by cholesterol. Our findings confirm the specific role of cholesterol in endothelial cells and may have implications for cholesterol-dependent vascular pathologies. PMID:23162471

  19. Low temperature synthesis of seed mediated CuO bundle of nanowires, their structural characterisation and cholesterol detection.

    PubMed

    Ibupoto, Z H; Khun, K; Liu, X; Willander, M

    2013-10-01

    In this study, we have successfully synthesised CuO bundle of nanowires using simple, cheap and low temperature hydrothermal growth method. The growth parameters such as precursor concentration and time for duration of growth were optimised. The field emission scanning electron microscopy (FESEM) has demonstrated that the CuO bundles of nanowires are highly dense, uniform and perpendicularly oriented to the substrate. The high resolution transmission electron microscopy (HRTEM) has demonstrated that the CuO nanostructures consist of bundle of nanowires and their growth pattern is along the [010] direction. The X-ray diffraction (XRD) technique described that CuO bundle of nanowires possess the monoclinic crystal phase. The surface and chemical composition analyses were carried out with X-ray photoelectron spectroscopy (XPS) technique and the obtained results suggested the pure crystal state of CuO nanostructures. In addition, the CuO nanowires were used for the cholesterol sensing application by immobilising the cholesterol oxidase through electrostatic attraction. The infrared reflection absorption spectroscopy study has also revealed that CuO nanostructures are consisting of only CuO bonding and has also shown the possible interaction of cholesterol oxidase with the sharp edge surface of CuO bundle of nanowires. The proposed cholesterol sensor has demonstrated the wide range of detection of cholesterol with good sensitivity of 33.88±0.96 mV/decade. Moreover, the CuO bundle of nanowires based sensor electrode has revealed good repeatability, reproducibility, stability, selectivity and a fast response time of less than 10s. The cholesterol sensor based on the immobilised cholesterol oxidase has good potential applicability for the determination of cholesterol from the human serum and other biological samples.

  20. Oxygen biosensor based on bilirubin oxidase immobilized on a nanostructured gold electrode.

    PubMed

    Pita, Marcos; Gutierrez-Sanchez, Cristina; Toscano, Miguel D; Shleev, Sergey; De Lacey, Antonio L

    2013-12-01

    Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase. PMID:23973738

  1. Hordeum vulgare Seedlings Amine Oxidase

    PubMed Central

    Cogoni, Antonina; Piras, Carla; Farci, Raffaele; Melis, Antonello; Floris, Giovanni

    1990-01-01

    Although no amine oxidase could be detected in crude extracts, the enzyme has been purified to apparent homogeneity from Hordeum vulgare seedlings using ammonium sulfate precipitation and chromatography on DEAE cellulose, Hydroxylapatite, and Sephadex G200 columns. Gel filtration experiments indicate a molecular weight of about 150,000. The pH optimum of the enzyme was found to be 7.5 in potassium phosphate buffer. The spectrum of ultraviolet and visible regions were similar to Cuamine oxidase from Leguminosae. PMID:16667542

  2. Fabrication of nanocrystalline CdS electrode via chemical bath deposition technique for application to cholesterol sensor

    NASA Astrophysics Data System (ADS)

    Dhyani, Hemant; Srivastava, Saurabh; Azahar Ali, Md; Malhotra, B. D.; Sen, Prasenjit

    2012-04-01

    A nanocystalline CdS electrode has been fabricated by chemical bath deposition (CBD) technique onto hydrolyzed indium tin oxide (ITO) coated glass substrate at 78°C for the immobilization of cholesterol oxidase (ChOx). The prepared Nano-CdS based electrode has been characterized using UV-visible, X-ray diffraction (XRD), Fourier transform-infrared (FTIR) and scanning electron microscopy (SEM). The ChOx/Nano-CdS/ITO bioelectrode shows the detection range of cholesterol from 50 to 400 mg/dl with improved sensitivity of 1.35 μA/mgdl-1/cm2, low detection limit (6.1 mg/dl) and low Km (0.45mM) value indicating strong enzyme (cholesterol oxidase)-matrix (CdS) affinity.

  3. Electrochemiluminescence imaging for parallel single-cell analysis of active membrane cholesterol.

    PubMed

    Zhou, Junyu; Ma, Guangzhong; Chen, Yun; Fang, Danjun; Jiang, Dechen; Chen, Hong-Yuan

    2015-08-18

    Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

  4. Effect of clofibrate on cholesterol metabolism in rats treated with polychlorinated biphenyls

    SciTech Connect

    Nakagawa, M.; Shimokawa, T.; Noguchi, A.; Ishihara, N.; Kojima, S.

    1986-02-01

    Serum and hepatic cholesterol content in rats treated with polychlorinated biphenyls (PCBs, KC-400) were increased compared to those of control rats. This increase of cholesterol content was reduced to control level by simultaneous administration of ethyl p-chlorophenoxyisobutyrate (CPIB). Also, when lecithin-cholesterol acyltransferase (LCAT) activity was expressed as the net cholesterol esterification, the acyltransferase activity in rats treated with PCBs was elevated, while the elevated acyltransferase activity was brought to control level by simultaneous administration of CPIB. On the other hand, the amount of bile of rats treated with CPIB, PCBs and PCBs-CPIB was increased, but free and total cholesterol content in bile of these treated rats was decreased to 40-60% of those of control rats. Moreover, cytochrome P-450 content in liver microsomes of rats treated with CPIB, PCBs and PCBs-CPIB was increased. At the same time, cholesterol-metabolizing activity in liver microsomes of rats treated with CPIB, PCBs and PCBs-CPIB also was elevated. Similar results were obtained for drug metabolizing (aniline hydroxylation and aminopyrine N-demethylation) activity. In addition, the amount of bile acids excreted from rats treated with CPIB, PCBs and PCBs-CPIB was increased compared to that of control rats. These results suggest that hypercholesterolemia induced by oral ingestion of PCBs is recovered by CPIB treatment and that this hypocholesterolemic effect of CPIB may be related partly to the elevation of hepatic mixed function oxidase activity for cholesterol catabolism.

  5. Expression of alternative oxidase in tomato

    SciTech Connect

    Kakefuda, M.; McIntosh, L. )

    1990-05-01

    Tomato fruit ripening is characterized by an increase in ethylene biosynthesis, a burst in respiration (i.e. the climacteric), fruit softening and pigmentation. As whole tomatoes ripened from mature green to red, there was an increase in the alternative oxidase capacity. Aging pink tomato slices for 24 and 48 hrs also showed an increase of alternative oxidase and cytochrome oxidase capacities. Monoclonal antibodies prepared to the Sauromatum guttatum alternative oxidase were used to follow the appearance of alternative oxidase in tomato fruits. There is a corresponding increase in a 36kDa protein with an increase in alternative oxidase capacity. Effects of ethylene and norbornadiene on alternative oxidase capacity were also studied. We are using an alternative oxidase cDNA clone from potato to study the expression of mRNA in ripening and wounded tomatoes to determine if the gene is transcriptionally regulated.

  6. An enzyme thermistor-based assay for total and free cholesterol.

    PubMed

    Raghavan, V; Ramanathan, K; Sundaram, P V; Danielsson, B

    1999-11-01

    A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit. PMID:10556661

  7. Cholesterol - what to ask your doctor

    MedlinePlus

    ... this page: //medlineplus.gov/ency/patientinstructions/000211.htm Cholesterol - what to ask your doctor To use the ... this page, please enable JavaScript. Your body needs cholesterol to work properly. When you have extra cholesterol ...

  8. What Do My Cholesterol Levels Mean?

    MedlinePlus

    ... Tools & Resources Stroke More What Do My Cholesterol Levels Mean? Updated:Mar 22,2016 High cholesterol can ... a fasting “lipoprotein profile” to measure your cholesterol levels. It assesses several types of fat in the ...

  9. How to Get Your Cholesterol Tested

    MedlinePlus

    ... HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and triglycerides. A small sample of blood will be drawn ... the amount of LDL (bad) cholesterol level and triglycerides can be affected by what you've recently ...

  10. Lateral organization of cholesterol molecules in lipid-cholesterol assemblies.

    SciTech Connect

    Singh, Rajiv R. P.; Slepoy, Alexander; Sengupta, Pinaki; Cox, Daniel L.

    2005-05-01

    We present results of an off-lattice simulation of a two-component planar system, as a model for lateral organization of cholesterol molecules in lipid-cholesterol assemblies. We explore the existence of 'superlattice' structures even in fluid systems, in the absence of an underlying translational long-range order, and study their coupling to hexatic or bond-orientational order. We discuss our results in context of geometric superlattice theories and 'condensation complexes' in understanding a variety of experiments in artificial lipid-cholesterol assemblies.

  11. Think Again About Cholesterol Survey.

    PubMed

    Catapano, Alberico L; Wiklund, Olov

    2015-12-01

    Cardiovascular disease (CVD) is still the main cause of death in Europe. Elevated plasma cholesterol, specifically low-density lipoprotein cholesterol (LDL-C), is the main causative risk factor for CVD, most prominently associated with coronary heart disease. A widespread disinformation about cholesterol and CVD is one factor underlying a poor compliance to lipid-lowering therapy. To investigate how cholesterol, CVD and cholesterol reduction is perceived in the population, a survey was commissioned by the European Atherosclerosis Society (EAS). Nearly half of people above 25 years of age are most worried about cancer (45%), compared to just over one in four who are worried about heart disease (27%). A majority believe being overweight (72%), blood pressure (70%) and smoking (67%) most affect heart health, far more than note cholesterol (59%) and family history (39%). The majority of adults recognize that high LDL (or "bad") cholesterol should be a health priority for everyone, including those younger than 40 and those who are not overweight. However, 1 in 4 (25%) incorrectly believe that it does not need to be a concern until someone shows signs or symptoms. Although 89% of adults surveyed agreed it is important for people to know whether or not they have high LDL-C, an overwhelming 92% did not know their LDL-C levels or had never had their cholesterol levels tested. A high 63% had never heard of familial hypercholesterolemia: France had the lowest level of awareness (41%) to Denmark with a high 80%, and the association of the disease with high levels of LDL-C is quite poor (only 36%), with Sweden only at 22% versus a high in Spain of 54%. A large part of the people participating in the survey were quite uncertain about the modality of transmission for familial hypercholesterolemia in the family. All in all, this survey highlights the need for more information among citizens for the role of cholesterol in determining CVD.

  12. Think Again About Cholesterol Survey.

    PubMed

    Catapano, Alberico L; Wiklund, Olov

    2015-12-01

    Cardiovascular disease (CVD) is still the main cause of death in Europe. Elevated plasma cholesterol, specifically low-density lipoprotein cholesterol (LDL-C), is the main causative risk factor for CVD, most prominently associated with coronary heart disease. A widespread disinformation about cholesterol and CVD is one factor underlying a poor compliance to lipid-lowering therapy. To investigate how cholesterol, CVD and cholesterol reduction is perceived in the population, a survey was commissioned by the European Atherosclerosis Society (EAS). Nearly half of people above 25 years of age are most worried about cancer (45%), compared to just over one in four who are worried about heart disease (27%). A majority believe being overweight (72%), blood pressure (70%) and smoking (67%) most affect heart health, far more than note cholesterol (59%) and family history (39%). The majority of adults recognize that high LDL (or "bad") cholesterol should be a health priority for everyone, including those younger than 40 and those who are not overweight. However, 1 in 4 (25%) incorrectly believe that it does not need to be a concern until someone shows signs or symptoms. Although 89% of adults surveyed agreed it is important for people to know whether or not they have high LDL-C, an overwhelming 92% did not know their LDL-C levels or had never had their cholesterol levels tested. A high 63% had never heard of familial hypercholesterolemia: France had the lowest level of awareness (41%) to Denmark with a high 80%, and the association of the disease with high levels of LDL-C is quite poor (only 36%), with Sweden only at 22% versus a high in Spain of 54%. A large part of the people participating in the survey were quite uncertain about the modality of transmission for familial hypercholesterolemia in the family. All in all, this survey highlights the need for more information among citizens for the role of cholesterol in determining CVD. PMID:26671304

  13. NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology

    PubMed Central

    Cave, Alison; Grieve, David; Johar, Sofian; Zhang, Min; Shah, Ajay M

    2005-01-01

    Chronic heart failure, secondary to left ventricular hypertrophy or myocardial infarction, is a condition with increasing morbidity and mortality. Although the mechanisms underlying the development and progression of this condition remain a subject of intense interest, there is now growing evidence that redox-sensitive pathways play an important role. This article focuses on the involvement of reactive oxygen species derived from a family of superoxide-generating enzymes, termed NADPH oxidases (NOXs), in the pathophysiology of ventricular hypertrophy, the accompanying interstitial fibrosis and subsequent heart failure. In particular, the apparent ability of the different NADPH oxidase isoforms to define the response of a cell to a range of physiological and pathophysiological stimuli is reviewed. If confirmed, these data would suggest that independently targeting different members of the NOX family may hold the potential for therapeutic intervention in the treatment of cardiac disease. PMID:16321803

  14. Cholesterol Perturbs Lipid Bilayers Nonuniversally

    SciTech Connect

    Pan Jianjun; Mills, Thalia T.; Tristram-Nagle, Stephanie; Nagle, John F.

    2008-05-16

    Cholesterol is well known to modulate the physical properties of biomembranes. Using modern x-ray scattering methods, we have studied the effects of cholesterol on the bending modulus K{sub C}, the thickness D{sub HH}, and the orientational order parameter S{sub xray} of lipid bilayers. We find that the effects are different for at least three classes of phospholipids characterized by different numbers of saturated hydrocarbon chains. Most strikingly, cholesterol strongly increases K{sub C} when both chains of the phospholipid are fully saturated but not at all when there are two monounsaturated chains.

  15. Targeting cancer using cholesterol conjugates

    PubMed Central

    Radwan, Awwad A.; Alanazi, Fares K.

    2013-01-01

    Conjugation of cholesterol moiety to active compounds for either cancer treatment or diagnosis is an attractive approach. Cholesterol derivatives are widely studied as cancer diagnostic agents and as anticancer derivatives either in vitro or in vivo using animal models. In largely growing studies, anticancer agents have been chemically conjugated to cholesterol molecules, to enhance their pharmacokinetic behavior, cellular uptake, target specificity, and safety. To efficiently deliver anticancer agents to the target cells and tissues, many different cholesterol–anticancer conjugates were synthesized and characterized, and their anticancer efficiencies were tested in vitro and in vivo. PMID:24493968

  16. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

  17. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    PubMed

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

  18. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

    PubMed Central

    Molitor, Christian; Mauracher, Stephan Gerhard

    2016-01-01

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

  19. Cholesterol confusion and statin controversy

    PubMed Central

    DuBroff, Robert; de Lorgeril, Michel

    2015-01-01

    The role of blood cholesterol levels in coronary heart disease (CHD) and the true effect of cholesterol-lowering statin drugs are debatable. In particular, whether statins actually decrease cardiac mortality and increase life expectancy is controversial. Concurrently, the Mediterranean diet model has been shown to prolong life and reduce the risk of diabetes, cancer, and CHD. We herein review current data related to both statins and the Mediterranean diet. We conclude that the expectation that CHD could be prevented or eliminated by simply reducing cholesterol appears unfounded. On the contrary, we should acknowledge the inconsistencies of the cholesterol theory and recognize the proven benefits of a healthy lifestyle incorporating a Mediterranean diet to prevent CHD. PMID:26225201

  20. Cholesterol's location in lipid bilayers

    DOE PAGESBeta

    Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R.; Harroun, Thad A.; Katsaras, John

    2016-04-04

    It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown – at least in some bilayers – to align differently from its canonical upright orientation, where its hydroxyl group is in themore » vicinity of the lipid–water interface. In this study we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies.« less

  1. Formation of cholesterol bilayer domains precedes formation of cholesterol crystals in cholesterol/dimyristoylphosphatidylcholine membranes: EPR and DSC studies.

    PubMed

    Mainali, Laxman; Raguz, Marija; Subczynski, Witold K

    2013-08-01

    Saturation-recovery EPR along with DSC were used to determine the cholesterol content at which pure cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in dimyristoylphosphatidylcholine (DMPC) membranes. To preserve compositional homogeneity throughout the membrane suspension, lipid multilamellar dispersions were prepared using a rapid solvent exchange method. The cholesterol content increased from 0 to 75 mol %. With spin-labeled cholesterol analogues, it was shown that the CBDs begin to form at ~50 mol % cholesterol. It was confirmed by DSC that the cholesterol solubility threshold for DMPC membranes is detected at ~66 mol % cholesterol. At levels above this cholesterol content, monohydrate cholesterol crystals start to form. The major finding is that the formation of CBDs precedes formation of cholesterol crystals. The region of the phase diagram for cholesterol contents between 50 and 66 mol % is described as a structured one-phase region in which CBDs have to be supported by the surrounding DMPC bilayer saturated with cholesterol. Thus, the phase boundary located at 66 mol % cholesterol separates the structured one-phase region (liquid-ordered phase of DMPC with CBDs) from the two-phase region where the structured liquid-ordered phase of DMPC coexists with cholesterol crystals. It is likely that CBDs are precursors of monohydrate cholesterol crystals.

  2. Cholesterol-dependent cytolysins.

    PubMed

    Gilbert, Robert J C

    2010-01-01

    The cholesterol-dependent cytolysins (CDCs) are part of a large family of pore-forming proteins that include the human proteins perforin and the complement membrane attack complex. The activity of all family members is focused on membranes, but the proteins are themselves involved in a diverse range of phenomena. An overview of some of these phenomena is provided here, along with an historical perspective of CDCs themselves and how our understanding of their mechanism of action has developed over the years. The way in which pore formation depends on specific characteristics of the membrane under attack as well as of the protein doing the attacking is emphasised. The cholesterol-dependent cytolysins (CDCs) have been the focus of a renewed keen research interest for over ten years now. Their importance has been even further enhanced by the homology now identified between them and the membrane attack complex/perforin (MACPF) family of proteins, which includes several components of the complement cascade as well as perforin itself. In this chapter I aim to provide an overview of our understanding of the interaction between CDCs and other members of what is now called the MACPF/CDC superfamily, with their target membranes. CDCs (also in the past known as thiol-activated toxins or cholesterol-binding toxins) were originally identified from four Gram-positive bacterial genera (Clostridium, Listeria, Bacillus and Streptococcus). Well-known examples include listeriolysin, perfringolysin, streptolysin and pneumoysin. Listeriolysin from L. monocytogenes is responsible for the escape of bacteria from the phagosome to colonise the cytoplasm and has been applied as a protein adjuvant in the development of vaccines against cancer and tuberculosis, for example. Perfringolysin from C. perfringens (Fig. 1A) has become perhaps the most studied CDC4 and has an important role in pathology associated with infection (gangrene). Streptolysin from S. pyogenes is another intensely studied

  3. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    PubMed

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  4. Facts about...Blood Cholesterol. Revised.

    ERIC Educational Resources Information Center

    National Heart, Lung, and Blood Inst. (DHHS/NIH), Bethesda, MD.

    This fact sheet on blood cholesterol examines the connection between cholesterol and heart disease, lists risk factors for heart disease that can and cannot be controlled, points out who can benefit from lowering blood cholesterol, distinguishes between blood and dietary cholesterol, describes low density lipoprotein and high density lipoprotein…

  5. Nanostructured anatase-titanium dioxide based platform for application to microfluidics cholesterol biosensor

    NASA Astrophysics Data System (ADS)

    Azahar Ali, Md.; Srivastava, Saurabh; Solanki, Pratima R.; Varun Agrawal, Ved; John, Renu; Malhotra, Bansi D.

    2012-08-01

    We report results of studies relating to the fabrication of a microfluidics cholesterol sensor based on nanocrystalline anatase-titanium dioxide (ant-TiO2) film deposited onto indium tin oxide (ITO) glass. The results of response studies (optimized under the flow rate of 30 μl/min) conducted on cholesterol oxidase (ChOx) immobilized onto crystalline ant-TiO2 nanoparticles (˜27 nm)/ITO microfluidics electrode reveal linearity as 1.3 to 10.3 mM and improved sensitivity of 94.65 μA/mM/cm2. The observed low value of Km (0.14 mM) indicates high affinity of ChOx to cholesterol. No significant changes in current response of this microfluidics sensor are measured in the presence of different interferents.

  6. Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film

    SciTech Connect

    Singh, S. P.; Arya, Sunil K.; Pandey, Pratibha; Malhotra, B. D.; Saha, Shibu; Sreenivas, K.; Gupta, Vinay

    2007-08-06

    Cholesterol oxidase (ChOx) has been immobilized onto zinc oxide (ZnO) nanoporous thin films grown on gold surface. A preferred c-axis oriented ZnO thin film with porous surface morphology has been fabricated by rf sputtering under high pressure. Optical studies and cyclic voltammetric measurements show that the ChOx/ZnO/Au bioelectrode is sensitive to the detection of cholesterol in 25-400 mg/dl range. A relatively low value of enzyme's kinetic parameter (Michaelis-Menten constant) {approx}2.1 mM indicates enhanced enzyme affinity of ChOx to cholesterol. The observed results show promising application of nanoporous ZnO thin film for biosensing application without any functionalization.

  7. Self assembled monolayer based liquid crystal biosensor for free cholesterol detection

    NASA Astrophysics Data System (ADS)

    Tyagi, Mukta; Chandran, Achu; Joshi, Tilak; Prakash, Jai; Agrawal, V. V.; Biradar, A. M.

    2014-04-01

    A unique cholesterol oxidase (ChOx) liquid crystal (LC) biosensor, based on the disruption of orientation in LCs, is developed for cholesterol detection. A self-assembled monolayer (SAM) of Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP) and (3-Aminopropyl)trimethoxy-silane (APTMS) is prepared on a glass plate by adsorption. The enzyme (ChOx) is immobilized on SAM surface for 12 h before utilizing the film for biosensing purpose. LC based biosensing study is conducted on SAM/ChOx/LC (5CB) cells for cholesterol concentrations ranging from 10 mg/dl to 250 mg/dl. The sensing mechanism has been verified through polarizing optical microscopy, scanning electron microscopy, and spectrometric techniques.

  8. Self assembled monolayer based liquid crystal biosensor for free cholesterol detection

    SciTech Connect

    Tyagi, Mukta; Agrawal, V. V.; Chandran, Achu; Joshi, Tilak; Prakash, Jai; Biradar, A. M.

    2014-04-14

    A unique cholesterol oxidase (ChOx) liquid crystal (LC) biosensor, based on the disruption of orientation in LCs, is developed for cholesterol detection. A self-assembled monolayer (SAM) of Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP) and (3-Aminopropyl)trimethoxy-silane (APTMS) is prepared on a glass plate by adsorption. The enzyme (ChOx) is immobilized on SAM surface for 12 h before utilizing the film for biosensing purpose. LC based biosensing study is conducted on SAM/ChOx/LC (5CB) cells for cholesterol concentrations ranging from 10 mg/dl to 250 mg/dl. The sensing mechanism has been verified through polarizing optical microscopy, scanning electron microscopy, and spectrometric techniques.

  9. Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes.

    PubMed

    Hayat, Akhtar; Haider, Waqar; Raza, Yousuf; Marty, Jean Louis

    2015-10-01

    A sensitive and selective colorimetric method based on the incorporation of zinc oxide nanoparticles (ZnO NPs) on the surface of carbon nanotubes (CNTs) was shown to posses synergistic peroxidase like activity for the detection of cholesterol. The proposed nanocomposite catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in the presence of hydrogen peroxide (H2O2) to produce a green colored product which can be monitored at 405 nm. H2O2 is the oxidative product of cholesterol in the presence of cholesterol oxidase. Therefore, the oxidation of cholesterol can be quantitatively related to the colorimetric response by combining these two reactions. Under the optimal experimental conditions, the colorimetric response was proportional to the concentration of cholesterol in the range of 0.5-500 nmol/L, with a detection limit of 0.2 nmol/L. The applicability of the proposed assays was demonstrated for the determination of cholesterol in milk powder samples with good recovery results. PMID:26078143

  10. Al:ZnO thin film: An efficient matrix for cholesterol detection

    NASA Astrophysics Data System (ADS)

    Batra, Neha; Tomar, Monika; Gupta, Vinay

    2012-12-01

    Al doped ZnO thin film (Al:ZnO) has been realized as a potential matrix for the development of efficient cholesterol biosensor. The correlation between the structural and electrical properties of ZnO thin film with varying Al doping concentration (1% to 5%) and their cyclic voltammetric (CV) response has been studied. 2% Al doped ZnO films were found to give the best CV response and were further utilized for immobilization of cholesterol oxidase (ChOx) to detect cholesterol. Amperometric and photometric studies reveal that the prepared bioelectrode based on 2% Al doped ZnO matrix (ChOx/Al:ZnO/Pt/glass) is highly sensitive (sensitivity = 173 μAmM-1 cm-2) to the detection of cholesterol in the wide range from 0.6-12.9 mM (25-500 mg/dl). A relatively low value of enzyme's kinetic parameter (Michaelis menten constant, 2.53 mM) indicates enhanced affinity of the immobilized ChOx toward cholesterol. The prepared bioelectrode is found to be exhibiting high shelf life (10 weeks) having negligible interference with the presence of other biomolecules in human serum indicating promising application of Al doped ZnO thin films for cholesterol biosensing.

  11. Colorimetric cholesterol sensor based on peroxidase like activity of zinc oxide nanoparticles incorporated carbon nanotubes.

    PubMed

    Hayat, Akhtar; Haider, Waqar; Raza, Yousuf; Marty, Jean Louis

    2015-10-01

    A sensitive and selective colorimetric method based on the incorporation of zinc oxide nanoparticles (ZnO NPs) on the surface of carbon nanotubes (CNTs) was shown to posses synergistic peroxidase like activity for the detection of cholesterol. The proposed nanocomposite catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in the presence of hydrogen peroxide (H2O2) to produce a green colored product which can be monitored at 405 nm. H2O2 is the oxidative product of cholesterol in the presence of cholesterol oxidase. Therefore, the oxidation of cholesterol can be quantitatively related to the colorimetric response by combining these two reactions. Under the optimal experimental conditions, the colorimetric response was proportional to the concentration of cholesterol in the range of 0.5-500 nmol/L, with a detection limit of 0.2 nmol/L. The applicability of the proposed assays was demonstrated for the determination of cholesterol in milk powder samples with good recovery results.

  12. Intestinal nuclear receptors in HDL cholesterol metabolism

    PubMed Central

    Degirolamo, Chiara; Sabbà, Carlo; Moschetta, Antonio

    2015-01-01

    The intestine plays a pivotal role in cholesterol homeostasis by functioning as an absorptive and secretory organ in the reverse cholesterol transport pathway. Enterocytes control cholesterol absorption, apoAI synthesis, HDL biogenesis, and nonbiliary cholesterol fecal disposal. Thus, intestine-based therapeutic interventions may hold promise in the management of diseases driven by cholesterol overload. Lipid-sensing nuclear receptors (NRs) are highly expressed in the intestinal epithelium and regulate transcriptionally the handling of cholesterol by the enterocytes. Here, we discuss the NR regulation of cholesterol fluxes across the enterocytes with special emphasis on NR exploitation as a bona fide novel HDL-raising strategy. PMID:25070952

  13. D1-like receptors regulate NADPH oxidase activity and subunit expression in lipid raft microdomains of renal proximal tubule cells.

    PubMed

    Li, Hewang; Han, Weixing; Villar, Van Anthony M; Keever, Lindsay B; Lu, Quansheng; Hopfer, Ulrich; Quinn, Mark T; Felder, Robin A; Jose, Pedro A; Yu, Peiying

    2009-06-01

    NADPH oxidase (Nox)-dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D(1)-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58+/-3.3%) than in hypertensive rats (31+/-5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67(phox) in cells from normotensive rats. D(1)-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-beta-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats. PMID:19380616

  14. [Role of HDL in Cholesterol Efflux and Reverse Cholesterol Transport].

    PubMed

    Ayaori, Makoto

    2016-01-01

    Low plasma levels of HDL-cholesterol (HDL-C) have been consistently associated with an increased risk of atherosclerotic cardiovascular diseases (CVD), and it is thus considered to be an anti-atherogenic lipoprotein. The development of novel therapies to enhance the atheroprotective properties of HDL may have the potential to further reduce the residual risk. Reverse cholesterol transport (RCT) is believed to be a primary atheroprotective property of HDL and its major protein, apolipoprotein A-I(apoA-I). HDL and apoA-I have been shown to promote the efflux of excess cholesterol from macrophage-derived foam cells via the cholesterol transporters, ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B, type I (SR-BI), and then transport it back to the liver for excretion into bile and eventually into the feces. In this regard, a validated murine assay that quantifies macrophage RCT may be a better predictor of atherosclerosis than the steady-state plasma concentration of HDL-C. Indeed, a recent clinical study demonstrated that the ability of serum HDL to mediate cholesterol efflux from macrophages was independently and negatively associated with the CVD risk even after adjustment for HDL-C levels, suggesting that HDL functionality is more important than its quantity. Therefore, the future development of HDL-targeted therapy should take both aspects into consideration to further reduce the residual risk.

  15. [Role of HDL in Cholesterol Efflux and Reverse Cholesterol Transport].

    PubMed

    Ayaori, Makoto

    2016-01-01

    Low plasma levels of HDL-cholesterol (HDL-C) have been consistently associated with an increased risk of atherosclerotic cardiovascular diseases (CVD), and it is thus considered to be an anti-atherogenic lipoprotein. The development of novel therapies to enhance the atheroprotective properties of HDL may have the potential to further reduce the residual risk. Reverse cholesterol transport (RCT) is believed to be a primary atheroprotective property of HDL and its major protein, apolipoprotein A-I(apoA-I). HDL and apoA-I have been shown to promote the efflux of excess cholesterol from macrophage-derived foam cells via the cholesterol transporters, ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B, type I (SR-BI), and then transport it back to the liver for excretion into bile and eventually into the feces. In this regard, a validated murine assay that quantifies macrophage RCT may be a better predictor of atherosclerosis than the steady-state plasma concentration of HDL-C. Indeed, a recent clinical study demonstrated that the ability of serum HDL to mediate cholesterol efflux from macrophages was independently and negatively associated with the CVD risk even after adjustment for HDL-C levels, suggesting that HDL functionality is more important than its quantity. Therefore, the future development of HDL-targeted therapy should take both aspects into consideration to further reduce the residual risk. PMID:27192798

  16. Heterogeneity of cholesterol homeostasis in man. Response to changes in dietary fat quality and cholesterol quantity.

    PubMed Central

    McNamara, D J; Kolb, R; Parker, T S; Batwin, H; Samuel, P; Brown, C D; Ahrens, E H

    1987-01-01

    Studies were carried out to examine the effects of dietary fat and cholesterol on cholesterol homeostasis in man. 75 12-wk studies were carried out during intake of 35% of calories as either saturated or polyunsaturated fat, first low and then high in dietary cholesterol. Dietary fat and cholesterol intakes, plasma lipid and lipoprotein levels, cholesterol absorption and sterol synthesis in isolated blood mononuclear leukocytes were measured during each diet period. In 69% of the studies the subjects compensated for the increased cholesterol intake by decreasing cholesterol fractional absorption and/or endogenous cholesterol synthesis. When an increase in plasma cholesterol levels was observed there was a failure to suppress endogenous cholesterol synthesis. Plasma cholesterol levels were more sensitive to dietary fat quality than to cholesterol quantity. The results demonstrate that the responses to dietary cholesterol and fat are highly individualized and that most individuals have effective feedback control mechanisms. PMID:3584466

  17. Phosphatidylcholine: Greasing the Cholesterol Transport Machinery

    PubMed Central

    Lagace, Thomas A.

    2015-01-01

    Negative feedback regulation of cholesterol metabolism in mammalian cells ensures a proper balance of cholesterol with other membrane lipids, principal among these being the major phospholipid phosphatidylcholine (PC). Processes such as cholesterol biosynthesis and efflux, cholesteryl ester storage in lipid droplets, and uptake of plasma lipoproteins are tuned to the cholesterol/PC ratio. Cholesterol-loaded macrophages in atherosclerotic lesions display increased PC biosynthesis that buffers against elevated cholesterol levels and may also facilitate cholesterol trafficking to enhance cholesterol sensing and efflux. These same mechanisms could play a generic role in homeostatic responses to acute changes in membrane free cholesterol levels. Here, I discuss the established and emerging roles of PC metabolism in promoting intracellular cholesterol trafficking and membrane lipid homeostasis. PMID:27081313

  18. Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

    PubMed

    Shoji, Atsushi; Ikeya, Kana; Aoyagi, Miki; Takatsuji, Ryutaro; Yanagida, Akio; Shibusawa, Yoichi; Sugawara, Masao

    2016-09-01

    Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the β-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanol<cholesterol<25-OH cholesterol and in a sterol concentration-dependent manner. The immobilization of liposomes consisting of PC, cholesterol and 4-cholesten-3-one exhibited a linear relationship between calcein permeability and the molar ratio of cholesterol and 4-cholesten-3-one. The SLO-based method was successfully applied for monitoring of cholesterol oxidase-mediated oxidation of cholesterol in a lipid bilayer. The potential of the SLO nanopore-based method for monitoring cholesterol oxidation in a lipid bilayer by other oxidative enzymes is also discussed.

  19. Female mice lacking active nadph-oxidase enzymes are protected against “western diet”--induced obesity and metabolic syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NADPH oxidase (Nox) enzymes have been implicated in regulation of adipocyte differentiation and inflammation in a variety of tissues. We examined the effects of feeding AIN-93G or a “Western diet” (WD) (45% fat, 0.5% cholesterol) on development of obesity and “metabolic syndrome” in wild type (WT) m...

  20. Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice.

    PubMed

    Schonewille, Marleen; Freark de Boer, Jan; Mele, Laura; Wolters, Henk; Bloks, Vincent W; Wolters, Justina C; Kuivenhoven, Jan A; Tietge, Uwe J F; Brufau, Gemma; Groen, Albert K

    2016-08-01

    Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we investigated the effects on cholesterol metabolism in mice in detail. Surprisingly, direct measurement of whole body cholesterol synthesis revealed that cholesterol synthesis was robustly increased in statin-treated mice. Measurement of organ-specific cholesterol synthesis demonstrated that the liver is predominantly responsible for the increase in cholesterol synthesis. Excess synthesized cholesterol did not accumulate in the plasma, as plasma cholesterol decreased. However, statin treatment led to an increase in cholesterol removal via the feces. Interestingly, enhanced cholesterol excretion in response to rosuvastatin and lovastatin treatment was mainly mediated via biliary cholesterol secretion, whereas atorvastatin mainly stimulated cholesterol removal via the transintestinal cholesterol excretion pathway. Moreover, we show that plasma cholesterol precursor levels do not reflect cholesterol synthesis rates during statin treatment in mice. In conclusion, cholesterol synthesis is paradoxically increased upon statin treatment in mice. However, statins potently stimulate the excretion of cholesterol from the body, which sheds new light on possible mechanisms underlying the cholesterol-lowering effects of statins.

  1. Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice.

    PubMed

    Schonewille, Marleen; Freark de Boer, Jan; Mele, Laura; Wolters, Henk; Bloks, Vincent W; Wolters, Justina C; Kuivenhoven, Jan A; Tietge, Uwe J F; Brufau, Gemma; Groen, Albert K

    2016-08-01

    Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we investigated the effects on cholesterol metabolism in mice in detail. Surprisingly, direct measurement of whole body cholesterol synthesis revealed that cholesterol synthesis was robustly increased in statin-treated mice. Measurement of organ-specific cholesterol synthesis demonstrated that the liver is predominantly responsible for the increase in cholesterol synthesis. Excess synthesized cholesterol did not accumulate in the plasma, as plasma cholesterol decreased. However, statin treatment led to an increase in cholesterol removal via the feces. Interestingly, enhanced cholesterol excretion in response to rosuvastatin and lovastatin treatment was mainly mediated via biliary cholesterol secretion, whereas atorvastatin mainly stimulated cholesterol removal via the transintestinal cholesterol excretion pathway. Moreover, we show that plasma cholesterol precursor levels do not reflect cholesterol synthesis rates during statin treatment in mice. In conclusion, cholesterol synthesis is paradoxically increased upon statin treatment in mice. However, statins potently stimulate the excretion of cholesterol from the body, which sheds new light on possible mechanisms underlying the cholesterol-lowering effects of statins. PMID:27313057

  2. Studies on serum LDL-cholesterol in second and third trimester of pregnancy.

    PubMed

    Husain, F; Latif, S A; Uddin, M M

    2010-07-01

    The present study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh, Bangladesh during the period of July 2006 to June 2007 to evaluate the effect of pregnancy on serum LDL-cholesterol. The serum concentrations of LDL-cholesterol was measured in 100cases during their 2nd and 3rd trimester of pregnancy and in a control group of 100 cases of non pregnant women which was matched on reproductive age. Data were analyzed by computer with SPSS program using unpaired student 't' test. The result showed that the pregnant women had significantly higher concentrations of serum LDL-cholesterol. Higher concentrations of serum LDL-cholesterol was more common in pregnant than control and reaching maximum at 3rd trimester of pregnancy. This may be a purely physiological response to pregnancy or it may be indicative of pathology in some women. These results warrant a follow up study to investigative whether the LDL hypercholesterolemia persists after parturition.

  3. Cholesterol as a co-solvent and a ligand for membrane proteins

    PubMed Central

    Song, Yuanli; Kenworthy, Anne K; Sanders, Charles R

    2014-01-01

    As of mid 2013 a Medline search on “cholesterol” yielded over 200,000 hits, reflecting the prominence of this lipid in numerous aspects of animal cell biology and physiology under conditions of health and disease. Aberrations in cholesterol homeostasis underlie both a number of rare genetic disorders and contribute to common sporadic and complex disorders including heart disease, stroke, type II diabetes, and Alzheimer's disease. The corresponding author of this review and his lab stumbled only recently into the sprawling area of cholesterol research when they discovered that the amyloid precursor protein (APP) binds cholesterol, a topic covered by the Hans Neurath Award lecture at the 2013 Protein Society Meeting. Here, we first provide a brief overview of cholesterol-protein interactions and then offer our perspective on how and why binding of cholesterol to APP and its C99 domain (β-CTF) promotes the amyloidogenic pathway, which is closely related to the etiology of Alzheimer's disease. PMID:24155031

  4. RADIOAUTOGRAPHY OF CHOLESTEROL IN LUNG

    PubMed Central

    Darrah, Hilary K.; Hedley-Whyte, John; Hedley-Whyte, E. Tessa

    1971-01-01

    30 Swiss albino mice aged 8 days were injected intraperitoneally with 0.2 ml of a solution of 4% N,N-dimethyl-formamide in 5% dextrose in water containing cholesterol-1,2-3H (∼1 mCi/ml). Lung tissue was embedded in an Epon mixture after either acetone and propylene oxide dehydration, partial ethanol and Epon 812 dehydration, or the precipitation of cholesterol by digitonin succeeded by partial dehydration. The distribution of cholesterol-1,2-3H in lung parenchyma in 1µ Epon section radioautograms was compared with that in frozen section radioautograms and was found to be independent of the manner of tissue processing. Grain distribution in the tissue was essentially the same whether 16, 63, 93, or 100% radioactivity was retained in the lung. However, grain distribution in the alveolar spaces differed, presumably due to displacement of pulmonary surfactant, which contains cholesterol. Intracellular distribution of cholesterol, in electron microscope radioautograms, was the same with either 51% or 93% retention of radioactivity in the lung. Loss of radioactivity into the various processing solutions was monitored. The various processing techniques have different drawbacks. PMID:19866763

  5. An amperometric bienzymatic cholesterol biosensor based on functionalized graphene modified electrode and its electrocatalytic activity towards total cholesterol determination.

    PubMed

    Manjunatha, Revanasiddappa; Shivappa Suresh, Gurukar; Melo, Jose Savio; D'Souza, Stanislaus F; Venkatesha, Thimmappa Venkatarangaiah

    2012-09-15

    Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been covalently immobilized onto functionalized graphene (FG) modified graphite electrode. Enzymes modified electrodes were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). FG accelerates the electron transfer from electrode surface to the immobilized ChOx, achieving the direct electrochemistry of ChOx. A well defined redox peak was observed, corresponding to the direct electron transfer of the FAD/FADH(2) of ChOx. The electron transfer coefficient (α) and electron transfer rate constant (K(s)) were calculated and their values are found to be 0.31 and 0.78 s(-1), respectively. For the free cholesterol determination, ChOx-FG/Gr electrode exhibits a sensitive response from 50 to 350 μM (R=-0.9972) with a detection limit of 5 μM. For total cholesterol determination, co-immobilization of ChEt and ChOx on modified electrode, i.e. (ChEt/ChOx)-FG/Gr electrode showed linear range from 50 to 300 μM (R=-0.9982) with a detection limit of 15 μM. Some common interferents like glucose, ascorbic acid and uric acid did not cause any interference, due to the use of a low operating potential. The FG/Gr electrode exhibits good electrocatalytic activity towards hydrogen peroxide (H(2)O(2)). A wide linear response to H(2)O(2) ranging from 0.5 to 7 mM (R=-0.9967) with a sensitivity of 443.25 μA mM(-1) cm(-2) has been obtained. PMID:22967556

  6. NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives.

    PubMed

    McCann, Sarah K; Roulston, Carli L

    2013-01-01

    Oxidative stress caused by an excess of reactive oxygen species (ROS) is known to contribute to stroke injury, particularly during reperfusion, and antioxidants targeting this process have resulted in improved outcomes experimentally. Unfortunately these improvements have not been successfully translated to the clinical setting. Targeting the source of oxidative stress may provide a superior therapeutic approach. The NADPH oxidases are a family of enzymes dedicated solely to ROS production and pre-clinical animal studies targeting NADPH oxidases have shown promising results. However there are multiple factors that need to be considered for future drug development: There are several homologues of the catalytic subunit of NADPH oxidase. All have differing physiological roles and may contribute differentially to oxidative damage after stroke. Additionally, the role of ROS in brain repair is largely unexplored, which should be taken into consideration when developing drugs that inhibit specific NADPH oxidases after injury. This article focuses on the current knowledge regarding NADPH oxidase after stroke including in vivo genetic and inhibitor studies. The caution required when interpreting reports of positive outcomes after NADPH oxidase inhibition is also discussed, as effects on long term recovery are yet to be investigated and are likely to affect successful clinical translation. PMID:24961415

  7. Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade.

    PubMed

    Kalachova, Tetiana; Iakovenko, Oksana; Kretinin, Sergii; Kravets, Volodymyr

    2013-05-01

    Salicylic acid is associated with the primary defense responses to biotic stress and formation of systemic acquired resistance. However, molecular mechanisms of early cell reactions to phytohormone application are currently undisclosed. The present study investigates the participation of phospholipase D and NADPH-oxidase in salicylic acid signal transduction cascade. The activation of lipid signaling enzymes within 15 min of salicylic acid application was shown in Arabidopsis thaliana plants by measuring the phosphatidic acid accumulation. Adding of primary alcohol (1-butanol) to the incubation medium led to phosphatidylbutanol accumulation as a result of phospholipase D (PLD) action in wild-type and NADPH-oxidase RbohD deficient plants. Salicylic acid induced rapid increase in NADPH-oxidase activity in histochemical assay with nitroblue tetrazolium but the reaction was not observed in presence of 1-butanol and NADPH-oxidase inhibitor diphenylene iodide (DPI). The further physiological effect of salicylic acid and inhibitory analysis of the signaling cascade were made in the guard cell model. Stomatal closure induced by salicylic acid was inhibited by 1-butanol and DPI treatment. rbohD transgenic plants showed impaired stomatal reaction upon phytohormone effect, while the reaction to H2O2 did not differ from that of wild-type plants. Thus a key role of NADPH-oxidase D-isoform in the process of stomatal closure in response to salicylic acid has been postulated. It has enabled to predict a cascade implication of PLD and NADPH oxidase to salicylic acid signaling pathway.

  8. Incorporation of copper into lysyl oxidase.

    PubMed

    Kosonen, T; Uriu-Hare, J Y; Clegg, M S; Keen, C L; Rucker, R B

    1997-10-01

    Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 microg/ml), glycosylation (tunicamycin, 10 microg/ml), protein secretion (brefeldin A, 10 microg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 microg/ml) together with 300 microCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Ci-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128-5134], copper is inserted into prolysyl oxidase independently of glycosylation. PMID:9355764

  9. The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.

    PubMed Central

    Carey, M C; Small, D M

    1978-01-01

    We determined the maximum solubilities of cholesterol in aqueous conjugated bile salt-egg lecithin-cholesterol systems as a function of several physical-chemical variables including those of physiological importance employing phase equilibria techniques. Equilibration rates are influenced by time and the method of sample preparation in that metastable supersaturation is readily induced at high bile salt: lecithin ratios, and equilibrium saturation by dissolution is achieved sluggisly at low bile salt:lecithin ratios. Equilibrium values for cholesterol saturation vary with the bile salt species, bile salt: lecithin ratio, temperature, ionic strength, and, in particular, with the total concentration of biliary lipids. Within physiological bile salt:lecithin ratios at 37 degreesC the influence of bile salt type and ionic strength is small, whereas the effects of bile salt:lecithin ratio and the total lipid concentration are major factors. We plotted on triangular coordinates a family of cholesterol solubility curves for each total lipid concentration (0.30--30 g/dl) and computed fifth-degree polynomial equations for each curve. With both the curves and the polynomial equations the "per cent cholesterol saturation" of fasting gallbladder and hepatic biles from patients with and without gallstones was calculated and both methods gave similar values. These results deomonstrate that by employing cholesterol saturation values appropriate to the total lipid concentration (range 0.2--24.9 g/dl) of individual biles, all cholesterol stone patients have supersaturated gallbladder biles, (mean, 132% [normal weight individuals], and 199% [morbidly obese individuals]). With controls and pigment stone patients the mean values were 95 and 98%, respectively, and in both approximately 50% of biles were supersaturated. Fasting hepatic biles were significantly more supersaturated than gallbladder biles (means 228--273%). Cholesterol monohydrate crystals were found in the majority of

  10. Polarizable multipolar electrostatics for cholesterol

    NASA Astrophysics Data System (ADS)

    Fletcher, Timothy L.; Popelier, Paul L. A.

    2016-08-01

    FFLUX is a novel force field under development for biomolecular modelling, and is based on topological atoms and the machine learning method kriging. Successful kriging models have been obtained for realistic electrostatics of amino acids, small peptides, and some carbohydrates but here, for the first time, we construct kriging models for a sizeable ligand of great importance, which is cholesterol. Cholesterol's mean total (internal) electrostatic energy prediction error amounts to 3.9 kJ mol-1, which pleasingly falls below the threshold of 1 kcal mol-1 often cited for accurate biomolecular modelling. We present a detailed analysis of the error distributions.

  11. Effect of ezetimibe on plasma cholesterol levels, cholesterol absorption, and secretion of biliary cholesterol in laboratory opossums with high and low responses to dietary cholesterol.

    PubMed

    Chan, Jeannie; Kushwaha, Rampratap S; Vandeberg, Jane F; Vandeberg, John L

    2008-12-01

    Partially inbred lines of laboratory opossums differ in plasma low-density lipoprotein cholesterol concentration and cholesterol absorption on a high-cholesterol diet. The aim of the present studies was to determine whether ezetimibe inhibits cholesterol absorption and eliminates the differences in plasma cholesterol and hepatic cholesterol metabolism between high and low responders on a high-cholesterol diet. Initially, we determined that the optimum dose of ezetimibe was 5 mg/(kg d) and treated 6 high- and 6 low-responding opossums with this dose (with equal numbers of controls) for 3 weeks while the opossums consumed a high-cholesterol and low-fat diet. Plasma and low-density lipoprotein cholesterol concentrations decreased significantly (P < .05) in treated but not in untreated high-responding opossums. Plasma cholesterol concentrations increased slightly (P < .05) in untreated low responders but not in treated low responders. The percentage of cholesterol absorption was significantly higher in untreated high responders than in other groups. Livers from high responders with or without treatment were significantly (P < .01) heavier than livers from low responders with or without treatment. Hepatic cholesterol concentrations in untreated high responders were significantly (P < .05) higher than those in low responders with or without treatment (P < .001). The gall bladder bile cholesterol concentrations in untreated high responders were significantly (P < .05) lower than those in other groups. A decrease in biliary cholesterol in low responders treated with ezetimibe was associated with a decrease in hepatic expression of ABCG5 and ABCG8. These studies suggest that ezetimibe decreases plasma cholesterol levels in high responders mainly by decreasing cholesterol absorption and increasing biliary cholesterol concentrations. Because ezetimibe's target is NPC1L1 and NPC1L1 is expressed in the intestine of opossums, its effect on cholesterol absorption may be mediated

  12. Physiological Waterfalls

    ERIC Educational Resources Information Center

    Leith, David E.

    1976-01-01

    Provides background information, defining areas within organ systems where physiological waterfalls exist. Describes pressure-flow relationships of elastic tubes (blood vessels, airways, renal tubules, various ducts). (CS)

  13. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  14. Glucose oxidase activity of actinomycetes.

    PubMed

    St Vlahov, S

    1978-01-01

    The ability of 311 actiomycete, belonging to 12 species to produce glucose oxidase was studied. It was found that 174 of them formed exoenzymes on solid medium and 133 in liquid medium. The composition of the nutrient medium has an essential effect on the amount of enzyme formed. Strains with considerably higher activity form a greater amount of exoenzymes on soya meal medium and on synthetic medium with KNO2. The highest activity of the culture liquid of some strains was observed between the 6th and 7th day of cultivation. During this phase of growth the highest productivity of the biomas was established. PMID:76424

  15. Nanomaterial-based Electrochemical Sensors for the Detection of Glucose and Cholesterol

    NASA Astrophysics Data System (ADS)

    Ahmadalinezhad, Asieh

    properties, we fabricated a highly sensitive and mediator-free electrochemical biosensor for the determination of total cholesterol. The developed biosensor possessed high selectivity and sensitivity (29.33 microA mM--1cm --2). The apparent Michaelis--Menten constant, KappM of this biosensor was very low (0.64 mM), which originated from both the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range, up to 300 mg dL--1 , in a physiological environment (pH 7.4); making it a promising candidate for the clinical determination of cholesterol. The fabricated biosensor was tested further by utilizing actual food samples (e.g., margarine, butter and fish oil). The results indicated that it has the potential capacity to be employed as a facile cholesterol detection tool in the food industry and for supplement quality control. To enhance the stability of the biosensors in the continuous monitoring of glucose, we designed a novel platform that was based on buckypaper. The fabricated biosensor responded to glucose with a considerable functional lifetime of over 80 days and detected glucose with a dynamic linear range of over 9 mM with a detection limit of 0.01 mM. To investigate the effects of the physical dimensions of nanomaterials on electrochemical biosensing, we synthesized TiO2 nanowires with controllable dimensions via a facile thermal oxidation treatment of a Ti substrate. To improve the conductivity of the TiO2 nanowires and to facilitate the immobilization of enzymes, a thin layer of carbon was deposited onto the TiO2 nanowires via a chemical vapour deposition method. Upon the immobilization of glucose oxidase as a model protein, direct electron transfer was observed in a mediator-free biosensing environment. Our electrochemical studies have revealed that the electron transfer rate of the immobilized glucose oxidase is strongly dependent on the dimensions of the carbonized TiO 2 nanowires, and that the

  16. Nanomaterial-based Electrochemical Sensors for the Detection of Glucose and Cholesterol

    NASA Astrophysics Data System (ADS)

    Ahmadalinezhad, Asieh

    properties, we fabricated a highly sensitive and mediator-free electrochemical biosensor for the determination of total cholesterol. The developed biosensor possessed high selectivity and sensitivity (29.33 microA mM--1cm --2). The apparent Michaelis--Menten constant, KappM of this biosensor was very low (0.64 mM), which originated from both the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range, up to 300 mg dL--1 , in a physiological environment (pH 7.4); making it a promising candidate for the clinical determination of cholesterol. The fabricated biosensor was tested further by utilizing actual food samples (e.g., margarine, butter and fish oil). The results indicated that it has the potential capacity to be employed as a facile cholesterol detection tool in the food industry and for supplement quality control. To enhance the stability of the biosensors in the continuous monitoring of glucose, we designed a novel platform that was based on buckypaper. The fabricated biosensor responded to glucose with a considerable functional lifetime of over 80 days and detected glucose with a dynamic linear range of over 9 mM with a detection limit of 0.01 mM. To investigate the effects of the physical dimensions of nanomaterials on electrochemical biosensing, we synthesized TiO2 nanowires with controllable dimensions via a facile thermal oxidation treatment of a Ti substrate. To improve the conductivity of the TiO2 nanowires and to facilitate the immobilization of enzymes, a thin layer of carbon was deposited onto the TiO2 nanowires via a chemical vapour deposition method. Upon the immobilization of glucose oxidase as a model protein, direct electron transfer was observed in a mediator-free biosensing environment. Our electrochemical studies have revealed that the electron transfer rate of the immobilized glucose oxidase is strongly dependent on the dimensions of the carbonized TiO 2 nanowires, and that the

  17. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

    PubMed Central

    Bokoch, G M; Prossnitz, V

    1992-01-01

    The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation. Images PMID:1310693

  18. Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane.

    PubMed

    Wüstner, Daniel; Modzel, Maciej; Lund, Frederik W; Lomholt, Michael A

    2016-09-01

    Cholesterol is an important lipid component of the plasma membrane (PM) of mammalian cells, where it is involved in control of many physiological processes, such as endocytosis, cell migration, cell signalling and surface ruffling. In an attempt to explain these functions of cholesterol, several models have been put forward about cholesterol's lateral and transbilayer organization in the PM. In this article, we review imaging techniques developed over the last two decades for assessing the distribution and dynamics of cholesterol in the PM of mammalian cells. Particular focus is on fluorescence techniques to study the lateral and inter-leaflet distribution of suitable cholesterol analogues in the PM of living cells. We describe also several methods for determining lateral cholesterol dynamics in the PM including fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT) and spot variation FCS coupled to stimulated emission depletion (STED) microscopy. For proper interpretation of such measurements, we provide some background in probe photophysics and diffusion phenomena occurring in cell membranes. In particular, we show the equivalence of the reaction-diffusion approach, as used in FRAP and FCS, and continuous time random walk (CTRW) models, as often invoked in SPT studies. We also discuss mass spectrometry (MS) based imaging of cholesterol in the PM of fixed cells and compare this method with fluorescence imaging of sterols. We conclude that evidence from many experimental techniques converges towards a model of a homogeneous distribution of cholesterol with largely free and unhindered diffusion in both leaflets of the PM. PMID:27016337

  19. Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase.

    PubMed

    Slotte, J P; Härmälä, A S; Jansson, C; Pörn, M I

    1990-12-14

    Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and

  20. Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase.

    PubMed

    Slotte, J P; Härmälä, A S; Jansson, C; Pörn, M I

    1990-12-14

    Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and

  1. Membrane cholesterol modulates cochlear electromechanics.

    PubMed

    Brownell, William E; Jacob, Stefan; Hakizimana, Pierre; Ulfendahl, Mats; Fridberger, Anders

    2011-06-01

    Changing the concentration of cholesterol in the plasma membrane of isolated outer hair cells modulates electromotility and prestin-associated charge movement, suggesting that a similar manipulation would alter cochlear mechanics. We examined cochlear function before and after depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) in an excised guinea pig temporal bone preparation. The mechanical response of the cochlear partition to acoustic and/or electrical stimulation was monitored using laser interferometry and time-resolved confocal microscopy. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents. Exposure to MβCD increased the magnitude and asymmetry of the response, without changing the frequency tuning of sound-evoked mechanical responses or cochlear microphonic potentials. Sodium salicylate reversibly blocked the enhanced electromechanical response in cholesterol depleted preparations. The increase of sound-evoked vibrations during positive current injection was enhanced following MβCD in some preparations. Imaging was used to assess cellular integrity which remained unchanged after several hours of exposure to MβCD in several preparations. The enhanced electromechanical response reflects an increase in outer hair cell electromotility and may reveal features of cholesterol distribution and trafficking in outer hair cells. PMID:21373862

  2. Membrane Cholesterol Modulates Superwarfarin Toxicity.

    PubMed

    Marangoni, M Natalia; Martynowycz, Michael W; Kuzmenko, Ivan; Braun, David; Polak, Paul E; Weinberg, Guy; Rubinstein, Israel; Gidalevitz, David; Feinstein, Douglas L

    2016-04-26

    Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content. PMID:27119638

  3. Early steps in steroidogenesis: intracellular cholesterol trafficking.

    PubMed

    Miller, Walter L; Bose, Himangshu S

    2011-12-01

    Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and "free" cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

  4. Cholesterol autoxidation in phospholipid membrane bilayers

    SciTech Connect

    Sevanian, A.; McLeod, L.L.

    1987-09-01

    Lipid peroxidation in unilamellar liposomes of known cholesterol-phospholipid composition was monitored under conditions of autoxidation or as induced by a superoxide radical generating system, gamma-irradiation or cumene hydroperoxide. Formation of cholesterol oxidation products was indexed to the level of lipid peroxidation. The major cholesterol oxidation products identified were 7-keto-cholesterol, isomeric cholesterol 5,6-epoxides, isomeric 7-hydroperoxides and isomeric 3,7-cholestane diols. Other commonly encountered products included 3,5-cholestadiene-7-one and cholestane-3 beta, 5 alpha, 6 beta-triol. Superoxide-dependent peroxidation required iron and produced a gradual increase in 7-keto-cholesterol and cholesterol epoxides. Cholesterol oxidation was greatest in liposomes containing high proportions of unsaturated phospholipid to cholesterol (4:1 molar ratio), intermediate with low phospholipid to cholesterol ratios (2:1) and least in liposomes prepared with dipalmitoylphosphatidylcholine and cholesterol. This relationship held regardless of the oxidizing conditions used. Cumene hydroperoxide-dependent lipid peroxidation and/or more prolonged oxidations with other oxidizing systems yielded a variety of products where cholesterol-5 beta,6 beta-epoxide, 7-ketocholesterol and the 7-hydroperoxides were most consistently elevated. Oxyradical initiation of lipid peroxidation produced a pattern of cholesterol oxidation products distinguishable from the pattern derived by cumene hydroperoxide-dependent peroxidation.

  5. Early steps in steroidogenesis: intracellular cholesterol trafficking

    PubMed Central

    Miller, Walter L.; Bose, Himangshu S.

    2011-01-01

    Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and “free” cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

  6. Rowing Physiology.

    ERIC Educational Resources Information Center

    Spinks, W. L.

    This review of the literature discusses and examines the methods used in physiological assessment of rowers, results of such assessments, and future directions emanating from research in the physiology of rowing. The first section discusses the energy demands of rowing, including the contribution of the energy system, anaerobic metabolism, and the…

  7. Topographic heterogeneity in cholesterol biosynthesis.

    PubMed

    Lange, Y; Muraski, M F

    1988-07-01

    We have examined the membrane topography of cholesterol biosynthesis in cultured human fibroblasts. We fed the cells with radioacetate and then interrupted the biosynthetic pathway so as to trap labeled intermediates in their subcellular locations. We analyzed homogenates of human fibroblasts labeled biosynthetically from radioacetate by centrifugation to equilibrium on sucrose gradients. The following two methods were used to interrupt cholesterol biosynthesis: incubation at 10 degrees C and treatment with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase. Incubation at 10 degrees C caused the accumulation of radiolanosterol at the expense of cholesterol. The lanosterol appeared predominantly at an unusually buoyant density (20% (w/w) sucrose; d = 1.08 g/cm3) as well as at the density normally labeled at 37 degrees C (30% sucrose; d = 1.13 g/cm3). 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol treatment caused the accumulation of labeled squalene and squalene 2,3-oxide. Reversal of the block permitted the label to progress rapidly as a wave into lanosterol and ultimately into cholesterol. The profiles of the three precursors did not coincide, suggesting that they were mostly in different membranes. Squalene was uniquely confined to a density of 1.18 g/cm3 (40% sucrose) while squalene 2,3-oxide appeared in peaks of density 1.08 g/cm3 and 1.13 g/cm3 (20% and 30% sucrose). Lanosterol was in a peak of density 1.13 g/cm3. Pulse-chase experiments showed that lanosterol synthesized in the membranes at 20% sucrose moved rapidly to the membranes at 30% sucrose where it was converted to cholesterol. The density gradient profiles of the following organelle markers also were monitored: plasma membrane, cholesterol mass; Golgi apparatus, galactosyltransferase; endoplasmic reticulum, RNA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cytochrome c reductase; peroxisomes, catalase. None of these markers appeared at the buoyant density

  8. Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase

    PubMed Central

    Byrne, Robert S.; Hänsch, Robert; Mendel, Ralf R.; Hille, Russ

    2009-01-01

    Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O2 and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O2] with a second-order rate constant of kox = 8.7 × 104 ± 0.5 × 104 m−1s−1. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed. PMID:19875441

  9. Immunological comparison of sulfite oxidase

    SciTech Connect

    Pollock, V.; Barber, M.J. )

    1991-03-11

    Polyclonal antibodies (rabbit), elicited against FPLC-purified chicken and rat liver sulfite oxidase (SO), have been examined for inhibition and binding to purified chicken (C), rat (R), bovine (B), alligator (A) and shark (S) liver enzymes. Anti-CSO IgG cross-reacted with all five enzymes, with varying affinities, in the order CSO=ASO{gt}RSO{gt}BSO{gt}SSO. Anti-ROS IgG also cross-reacted with all five enzymes in the order RSO{gt}CSO=ASO{gt}BSO{gt}SSO. Anti-CSO IgG inhibited sulfite:cyt. c reductase (S:CR), sulfite:ferricyanide reductase (S:FR) and sulfite:dichlorophenolindophenol reductase (S:DR) activities of CSO to different extents (S:CR{gt}S:FR=S:DR). Similar differential inhibition was found for anti-ROS IgG and RSO S:CR, S:FR and S:DR activities. Anti-CSO IgG inhibited S:CR activities in the order CSO=ASO{much gt}SSO{gt}BSO. RSO was uninhibited. For anti-RSO IgG the inhibition order was RSO{gt}SSO{gt}BSO{gt}ASO. CSO was uninhibited. Anti-CSO and RSO IgGs partially inhibited Chlorella nitrate reductase (NR). Minor cross-reactivity was found for xanthine oxidase. Common antigenic determinants for all five SO's and NR are indicated.

  10. Mitochondrial Cytochrome c Oxidase Deficiency

    PubMed Central

    Rak, Malgorzata; Bénit, Paule; Chrétien, Dominique; Bouchereau, Juliette; Schiff, Manuel; El-Khoury, Riyad; Tzagoloff, Alexander; Rustin, Pierre

    2016-01-01

    As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance to study different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy. PMID:26846578

  11. Double Potential Pulse Chronocoulometry for Detection of Plasma Membrane Cholesterol Efflux at Disk Platinum Microelectrodes

    PubMed Central

    West, Richard H.; Lu, Hui; Shaw, Kendrick; Chiel, Hillel J.; Kelley, Thomas J.; Burgess, James D.

    2016-01-01

    A double potential pulse scheme is reported for observation of cholesterol efflux from the plasma membrane of a single neuron cell. Capillary Pt disk microelectrodes having a thin glass insulator allow the 10 μm diameter electrode and cell to be viewed under optical magnification. The electrode, covalently functionalized with cholesterol oxidase, is positioned in contact with the cell surface resulting in enzyme catalyzed cholesterol oxidation and efflux of cholesterol from the plasma membrane at the electrode contact site. Enzymatically generated hydrogen peroxide accumulates at the electrode/cell interface during a 5 s hold-time and is oxidized during application of a potential pulse. A second, replicate potential pulse is applied 0.5 s after the first potential pulse to gauge background charge prior to significant accumulation of hydrogen peroxide. The difference in charge passed between the first and second potential pulse provides a measure of hydrogen peroxide generated by the enzyme and is an indication of the cholesterol efflux. Control experiments for bare Pt microelectrodes in contact with the cell plasma membrane show difference charge signals in the range of about 7–10 pC. Enzyme-modified electrodes in contact with the plasma membrane show signals in the range of 16–26 pC. PMID:27330196

  12. Inhibition of xanthine oxidase to prevent statin-induced myalgia and rhabdomiolysis.

    PubMed

    Alis, Rafael; Sanchis-Gomar, Fabian; Risso-Ballester, Jennifer; Perez-Quilis, Carme; Cortell-Ballester, Jose; Romagnoli, Marco; Blesa, Jose R; Emanuele, Enzo

    2015-03-01

    Although statins remain the cornerstone of lipid-lowering therapy for reducing the burden of atherosclerotic vascular disease, their administration has been associated with muscle-related adverse effects, including myalgia and rhabdomyolysis. Such adverse events are probably due to reduced antioxidant defenses associated with fewer intermediate metabolites in the cholesterol synthesis pathway. We hypothesize that the concomitant inhibition of xanthine oxidase via coadministration of allopurinol with statins could diminish reactive oxygen species (ROS)-related muscle damage, which would have in turn have positive effects on both the incidence of muscle-related adverse events and cardiovascular outcomes. Accordingly, inhibition of xanthine oxidase has been previously shown to be effective for reducing biomarkers of muscle damage following exercise in professional athletes. Because of the widespread statin utilization and increasing trends in their therapeutic use in atherosclerotic vascular diseases, the proposed strategy could have important clinical implications for reducing statin-induced myalgia and rhabdomyolysis. PMID:25568951

  13. Inherited Cholesterol Disorder Significantly Boosts Heart Risks

    MedlinePlus

    ... genetic disorder that causes high levels of "bad" LDL cholesterol have an increased risk for heart disease and ... in previous studies. Compared to people with average LDL cholesterol levels (less than 130 mg/dL), those with ...

  14. High Cholesterol: Medicines to Help You

    MedlinePlus

    ... Consumer Information by Audience For Women High Cholesterol--Medicines To Help You Share Tweet Linkedin Pin it ... Test to check your cholesterol (LDL-C) Combination Medicines Brand Name Generic Name Advicor Niacin and Lovastatin ...

  15. Do You Know Your Cholesterol Levels?

    MedlinePlus

    ... Health Information Center Do You Know Your Cholesterol Levels? Print-friendly Version (PDF, 6.1 MB) Spanish ... Syndrome? My Family Plan To Lower Blood Cholesterol Levels My Heart Health Card Play It Smart, Take ...

  16. Spatiotemporal Localization of d-Amino Acid Oxidase and d-Aspartate Oxidases during Development in Caenorhabditis elegans

    PubMed Central

    Saitoh, Yasuaki; Katane, Masumi; Kawata, Tomonori; Maeda, Kazuhiro; Sekine, Masae; Furuchi, Takemitsu; Kobuna, Hiroyuki; Sakamoto, Taro; Inoue, Takao; Arai, Hiroyuki; Nakagawa, Yasuhito

    2012-01-01

    Recent investigations have shown that a variety of d-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. d-Amino acid oxidase (DAO) and d-aspartate oxidase (DDO) are degradative enzymes stereospecific for d-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of d-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several d- and l-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and d-amino acids in multicellular organisms. PMID:22393259

  17. Quercetin regulates hepatic cholesterol metabolism by promoting cholesterol-to-bile acid conversion and cholesterol efflux in rats.

    PubMed

    Zhang, Min; Xie, Zongkai; Gao, Weina; Pu, Lingling; Wei, Jingyu; Guo, Changjiang

    2016-03-01

    Quercetin, a common member of the flavonoid family, is widely present in plant kingdom. Despite that quercetin is implicated in regulating cholesterol metabolism, the molecular mechanism is poorly understood. We hypothesized that quercetin regulates cholesterol homeostasis through regulating the key enzymes involved in hepatic cholesterol metabolism. To test this hypothesis, we compared the profile of key enzymes and transcription factors involved in the hepatic cholesterol metabolism in rats with or without quercetin supplementation. Twenty male Wistar rats were randomly divided into control and quercetin-supplemented groups. Serum total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and total bile acids in feces and bile were measured. Hepatic enzymatic activities were determined by activity assay kit and high-performance liquid chromatography-based analyses. The messenger RNA (mRNA) and protein expressions were determined by reverse transcriptase polymerase chain reaction and Western blot analyses, respectively. The results showed that the activity of hepatic cholesterol 7α-hydroxylase, a critical enzyme in the conversion of cholesterol to bile acids, was significantly elevated by quercetin. The expression of cholesterol 7α-hydroxylase, as well as liver X receptor α, an important transcription factor, was also increased at both mRNA and protein levels by quercetin. However, quercetin exposure had no impact on the activity of hepatic HMG-CoA reductase, a rate-limiting enzyme in the biosynthesis of cholesterol. We also found that quercetin treatment significantly increased ATP binding cassette transporter G1 mRNA and protein expression in the liver, suggesting that quercetin may increase hepatic cholesterol efflux. Collectively, the results presented here indicate that quercetin regulates hepatic cholesterol metabolism mainly through the pathways that promote cholesterol-to-bile acid conversion and

  18. Isolation of Cholesterol from an Egg Yolk

    ERIC Educational Resources Information Center

    Taber, Douglass F.; Li, Rui; Anson, Cory M.

    2011-01-01

    A simple procedure for the isolation of the cholesterol, by hydrolysis and extraction followed by column chromatography, is described. The cholesterol can be further purified by complexation with oxalic acid. It can also be oxidized and conjugated to cholestenone. The source of the cholesterol is one egg yolk, which contains about 200 mg of…

  19. Cholesterol Screening: A Practical Guide to Implementation.

    ERIC Educational Resources Information Center

    Kingery, Paul M.

    1995-01-01

    Dry-chemistry cholesterol analysis has made screening feasible in a variety of settings. The article provides practical tips for the implementation of mass cholesterol screening using a portable dry-chemistry analyzer and discusses issues involved in conducting effective cholesterol screening programs from start to finish. (SM)

  20. Non-cholesterol sterols and cholesterol metabolism in sitosterolemia.

    PubMed

    Othman, Rgia A; Myrie, Semone B; Jones, Peter J H

    2013-12-01

    Sitosterolemia (STSL) is a rare autosomal recessive disease, manifested by extremely elevated plant sterols (PS) in plasma and tissue, leading to xanthoma and premature atherosclerotic disease. Therapeutic approaches include limiting PS intake, interrupting enterohepatic circulation of bile acid using bile acid binding resins such as cholestyramine, and/or ileal bypass, and inhibiting intestinal sterol absorption by ezetimibe (EZE). The objective of this review is to evaluate sterol metabolism in STSL and the impact of the currently available treatments on sterol trafficking in this disease. The role of PS in initiation of xanthomas and premature atherosclerosis is also discussed. Blocking sterols absorption with EZE has revolutionized STSL patient treatment as it reduces circulating levels of non-cholesterol sterols in STSL. However, none of the available treatments including EZE have normalized plasma PS concentrations. Future studies are needed to: (i) explore where cholesterol and non-cholesterol sterols accumulate, (ii) assess to what extent these sterols in tissues can be mobilized after blocking their absorption, and (iii) define the factors governing sterol flux.

  1. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY -cholesterol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantifying cholesterol efflux involves labeling cells with [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]cholesterol is not ideal for...

  2. Percentage of Adults with High Cholesterol Whose LDL Cholesterol Levels Are Adequately Controlled

    MedlinePlus

    ... of Adults with High Cholesterol Whose LDL Cholesterol Levels are Adequately Controlled High cholesterol can double a ... with High Cholesterol that is Controlled by Education Level 8k4c-k22f Download these data » Click on legends ...

  3. Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments.

    PubMed

    Zhou, Guangqi; Yin, Jianhua; Chen, Haijiang; Hua, Yijie; Sun, Linlin; Gao, Haichun

    2013-09-01

    Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa3- and cbb3-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb3-type oxidase is the predominant system for respiration of oxygen (O2), especially when O2 is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb3-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa3-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb3- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa3-type oxidase and switch of the regulatory system for respiration.

  4. Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes.

    PubMed Central

    Samsonov, A V; Mihalyov, I; Cohen, F S

    2001-01-01

    Lipids segregate with each other into small domains in biological membranes, which can facilitate the associations of particular proteins. The segregation of cholesterol and sphingomyelin (SPM) into domains known as rafts is thought to be especially important. The formation of rafts was studied by using planar bilayer membranes that contained rhodamine-phosphatidylethanolamine (rho-DOPE) as a fluorescent probe, and wide-field fluorescence microscopy was used to detect phase separation of the probe. A fluorescently labeled GM(1), known to preferentially partition into rafts, verified that rho-DOPE faithfully reported the rafts. SPM-cholesterol domains did not form at high temperatures but spontaneously formed when temperature was lowered to below the melting temperature of the SPM. Saturated acyl chains on SPMs therefore promote the formation of rafts. The domains were circular (resolution > or = 0.5 microm), quickly reassumed their circular shape after they were deformed, and merged with each other to create larger domains, all phenomena consistent with liquid-ordered (l(o)) rather than solid-ordered (s(o)) domains. A saturated phosphatidylcholine (PC), disteoryl-PC, could substitute for SPM to complex with cholesterol into a l(o)-domain. But in the presence of cholesterol, a saturated phosphatidylethanolamine or phosphatidylserine yielded s(o)-domains of irregular shape. Lipids with saturated acyl chains can therefore pack well among each other and with cholesterol to form l(o)-domains, but domain formation is dependent on the polar headgroup of the lipid. An individual raft always extended through both monolayers. Degrading cholesterol in one monolayer with cholesterol oxidase first caused the boundary of the raft to become irregular; then the raft gradually disappeared. The fluid nature of rafts, demonstrated in this study, may be important for permitting dynamic interactions between proteins localized within rafts. PMID:11509362

  5. Anatomy & Physiology

    MedlinePlus

    ... Central Nervous System Peripheral Nervous System Review Quiz Endocrine System Characteristics of Hormones Endocrine Glands & Their Hormones Pituitary & ... Thyroid & Parathyroid Glands Adrenal Gland Pancreas Gonads Other Endocrine Glands ... Cardiovascular System Heart Structure of the Heart Physiology of the ...

  6. D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

    NASA Astrophysics Data System (ADS)

    Alfonso-García, Alba; Pfisterer, Simon G.; Riezman, Howard; Ikonen, Elina; Potma, Eric O.

    2016-06-01

    We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

  7. D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage.

    PubMed

    Alfonso-García, Alba; Pfisterer, Simon G; Riezman, Howard; Ikonen, Elina; Potma, Eric O

    2016-06-01

    We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

  8. Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study

    NASA Astrophysics Data System (ADS)

    Mandal, Pritam; Noutsi, Pakiza; Chaieb, Sahraoui

    2016-06-01

    Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells’ behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low.

  9. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    SciTech Connect

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S. )

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with ({sup 14}C)sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans.

  10. Fish protein hydrolysates affect cholesterol metabolism in rats fed non-cholesterol and high-cholesterol diets.

    PubMed

    Hosomi, Ryota; Fukunaga, Kenji; Arai, Hirofumi; Kanda, Seiji; Nishiyama, Toshimasa; Yoshida, Munehiro

    2012-03-01

    Fish consumption is well known to provide health benefits in both experimental animals and human subjects. Numerous studies have demonstrated the beneficial effects of various protein hydrolysates on lipid metabolism. In this context, this study examined the effect of fish protein hydrolysates (FPH) on cholesterol metabolism compared with the effect of casein. FPHs were prepared from Alaska pollock meat using papain as a protease. Male Wistar rats were divided into the following four dietary groups of seven rats each: either casein (20%) or FPH (10%) + casein (10%), with or without 0.5% cholesterol and 0.1% sodium cholate. Serum and liver lipid levels, fecal cholesterol and bile acid excretions, and the hepatic expression of genes encoding proteins involved in cholesterol homeostasis were examined. In rats fed the FPH diets compared with casein diets with or without cholesterol and sodium cholate, the indexes of cholesterol metabolism-namely, serum cholesterol, triglyceride, and low-density lipoprotein-cholesterol levels-were significantly lower, whereas fecal cholesterol and bile acid excretions were higher. Rats fed the FPH diets compared with casein with cholesterol exhibited a lower liver cholesterol level via an increased liver cholesterol 7α-hydroxylase (CYP7A1) expression level. This study demonstrates that the intake of FPH has hypocholesterolemic effects through the enhancement of fecal cholesterol and bile acid excretions and CYP7A1 expression levels. Therefore, fish peptides prepared by papain digestion might provide health benefits by decreasing the cholesterol content in the blood, which would contribute to the prevention of circulatory system diseases such as arteriosclerosis. PMID:22181072

  11. Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation.

    PubMed

    Smoak, Kathleen A; Aloor, Jim J; Madenspacher, Jennifer; Merrick, B Alex; Collins, Jennifer B; Zhu, Xuewei; Cavigiolio, Giorgio; Oda, Michael N; Parks, John S; Fessler, Michael B

    2010-06-01

    Crosstalk exists in mammalian cells between cholesterol trafficking and innate immune signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces antiatherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is proatherogenic. However, whether innate immunity plays an endogenous, physiological role in host cholesterol homeostasis in the absence of infection is undetermined. We report that apoA-I signals in the macrophage through Toll-like receptor (TLR)2, TLR4, and CD14, utilizing myeloid differentiation primary response protein 88 (MyD88)-dependent and -independent pathways, to activate nuclear factor-kappaB and induce cytokines. MyD88 plays a critical role in reverse cholesterol transport in vitro and in vivo, in part through promoting ATP-binding cassette A1 transporter upregulation. Taken together, this work identifies apoA-I as an endogenous stimulus of innate immunity that couples cholesterol trafficking to inflammation through MyD88 and identifies innate immunity as a physiologic signal in cholesterol homeostasis.

  12. Production of the carbonate radical anion during xanthine oxidase turnover in the presence of bicarbonate.

    PubMed

    Bonini, Marcelo G; Miyamoto, Sayuri; Di Mascio, Paolo; Augusto, Ohara

    2004-12-10

    Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants. PMID:15448145

  13. Expression of NADPH oxidase by trophoblast cells: potential implications for the postimplanting mouse embryo.

    PubMed

    Gomes, Sara Z; Lorenzon, Aline R; Vieira, Juliana S; Rocha, Clarissa R R; Bandeira, Clara; Hoshida, Mara S; Lopes, Lucia Rosseti; Bevilacqua, Estela

    2012-02-01

    Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses. PMID:22075471

  14. Peptide mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K.; Johansson, Jan

    2013-04-09

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  15. Does cholesterol lowering prevent stroke?

    PubMed

    Henry, R Y; Kendall, M J

    1998-10-01

    The importance of lowering plasma cholesterol to reduce the incidence of coronary events is well established. However, in the prevention of stroke disease, control of hypertension has been the main aim of treatment and lipid lowering therapy has not hitherto been considered to be desirable or necessary. In this review, the evidence from large multicentre trials, imaging studies and meta-analyses is presented. It shows convincingly that HMG-CoA reductase inhibitors (Statins) reduce stroke risk. PMID:9875681

  16. Structural Insights into Sulfite Oxidase Deficiency

    SciTech Connect

    Karakas,E.; Wilson, H.; Graf, T.; Xiang, S.; Jaramillo-Busquets, S.; Rajagopalan, K.; Kisker, C.

    2005-01-01

    Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

  17. Human lysyl oxidase-like 2.

    PubMed

    Moon, Hee-Jung; Finney, Joel; Ronnebaum, Trey; Mure, Minae

    2014-12-01

    Lysyl oxidase like-2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises Cu(2+)- and lysine tyrosylquinone (LTQ)-dependent amine oxidases. LOXL2 is proposed to function similarly to LOX in the extracellular matrix (ECM) by promoting crosslinking of collagen and elastin. LOXL2 has also been proposed to regulate extracellular and intracellular cell signaling pathways. Dysregulation of LOXL2 has been linked to many diseases, including cancer, pro-oncogenic angiogenesis, fibrosis and heart diseases. In this review, we will give an overview of the current understandings and hypotheses regarding the molecular functions of LOXL2.

  18. Molecular Mechanisms Underlying the Link between Nuclear Receptor Function and Cholesterol Gallstone Formation

    PubMed Central

    Vázquez, Mary Carmen; Rigotti, Attilio; Zanlungo, Silvana

    2012-01-01

    Cholesterol gallstone disease is highly prevalent in western countries, particularly in women and some specific ethnic groups. The formation of water-insoluble cholesterol crystals is due to a misbalance between the three major lipids present in the bile: cholesterol, bile salts, and phospholipids. Many proteins implicated in biliary lipid secretion in the liver are regulated by several transcription factors, including nuclear receptors LXR and FXR. Human and murine genetic, physiological, pathophysiological, and pharmacological evidence is consistent with the relevance of these nuclear receptors in gallstone formation. In addition, there is emerging data that also suggests a role for estrogen receptor ESR1 in abnormal cholesterol metabolism leading to gallstone disease. A better comprehension of the role of nuclear receptor function in gallstone formation may help to design new and more effective therapeutic strategies for this highly prevalent disease condition. PMID:22132343

  19. Liver X Receptor β and Peroxisome Proliferator-Activated Receptor δ regulate cholesterol transport in cholangiocytes

    PubMed Central

    Xia, Xuefeng; Jung, Dongju; Webb, Paul; Zhang, Aijun; Zhang, Bin; Li, Lifei; Ayers, Stephen D.; Gabbi, Chiara; Ueno, Yoshiyuki; Gustafsson, Jan-Åke; Alpini, Gianfranco; Moore, David D.; LeSage, Gene D.

    2012-01-01

    Nuclear receptors (NRs) play crucial roles in regulation of hepatic cholesterol synthesis, metabolism and conversion to bile acids, but their actions in cholangiocytes have not been examined. In this study, we investigated the roles of NRs in cholangiocyte physiology and cholesterol metabolism and flux. We examined the expression of NRs and other genes involved in cholesterol homeostasis in freshly isolated and cultured rodent cholangiocytes and found that these cells express a specific subset of NRs which includes Liver X Receptor β (LXRβ) and Peroxisome Proliferator-Activated Receptor δ (PPARδ). Activation of LXRβ and/or PPARδ in cholangiocytes induces ATP-binding cassette cholesterol transporter A1 (ABCA1) and increases cholesterol export at the basolateral compartment in polarized cultured cholangiocytes. In addition, PPARδ induces Niemann Pick C1 Like L1 (NPC1L1), which imports cholesterol into cholangiocytes and is expressed on the apical cholangiocyte membrane, via specific interaction with a PPRE within the NPC1L1 promoter. Based on these studies, we propose that (i) LXRβ and PPARδ coordinate NPC1L1/ABCA1 dependent vectorial cholesterol flux from bile through cholangiocytes and (ii) manipulation of these processes may influence bile composition with important applications in cholestatic liver disease and gallstone disease, serious health concerns for humans. PMID:22729460

  20. Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2[S

    PubMed Central

    Oninla, Vincent O.; Breiden, Bernadette; Babalola, Jonathan O.; Sandhoff, Konrad

    2014-01-01

    During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. PMID:25339683

  1. Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

    PubMed

    Oninla, Vincent O; Breiden, Bernadette; Babalola, Jonathan O; Sandhoff, Konrad

    2014-12-01

    During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.

  2. Thyroid hormone induction of human cholesterol 7 alpha-hydroxylase (Cyp7a1) in vitro

    PubMed Central

    Lammel Lindemann, Jan A.; Angajala, Anusha; Engler, David A.; Webb, Paul; Ayers, Stephen D.

    2014-01-01

    Thyroid hormone (TH) modulates serum cholesterol by acting on TH receptor β1 (TRβ1) in liver to regulate metabolic gene sets. In rodents, one important TH regulated step involves induction of Cyp7a1, an enzyme in the cytochrome P450 family, which enhances cholesterol to bile acid conversion and plays a crucial role in regulation of serum cholesterol levels. Current models suggest, however, that Cyp7a1 has lost the capacity to respond to THs in humans. We were prompted to re-examine TH effects on cholesterol metabolic genes in human liver cells by a recent study of a synthetic TH mimetic which showed that serum cholesterol reductions were accompanied by increases in a marker for bile acid synthesis in humans. Here, we show that TH effects upon cholesterol metabolic genes are almost identical in mouse liver, mouse and human liver primary cells and human hepatocyte cell lines. Moreover, Cyp7a1 is a direct TR target gene that responds to physiologic TR levels through a set of distinct response elements in its promoter. These findings suggest that THs regulate cholesterol to bile acid conversion in similar ways in humans and rodent experimental models and that manipulation of hormone signaling pathways could provide a strategy to enhance Cyp7a1 activity in human patients. PMID:24582860

  3. Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

    PubMed

    Oninla, Vincent O; Breiden, Bernadette; Babalola, Jonathan O; Sandhoff, Konrad

    2014-12-01

    During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. PMID:25339683

  4. Regulation of biliary cholesterol secretion in the rat. Role of hepatic cholesterol esterification.

    PubMed Central

    Nervi, F; Bronfman, M; Allalón, W; Depiereux, E; Del Pozo, R

    1984-01-01

    Although the significance of the enterohepatic circulation of bile salts in the solubilization and biliary excretion of cholesterol is well established, little is known about the intrahepatic determinants of biliary cholesterol output. Studies were undertaken to elucidate some of these determinants in the rat. Feeding 1% diosgenin for 1 wk increased biliary cholesterol output and saturation by 400%. Bile flow, biliary bile salt, phospholipid and protein outputs remained in the normal range. When ethynyl estradiol (EE) was injected into these animals, biliary cholesterol output decreased to almost normal levels under circumstances of minor changes in the rates of biliary bile salt and phospholipid outputs. Similarly, when chylomicron cholesterol was intravenously injected into diosgenin-fed animals, biliary cholesterol output significantly decreased as a function of the dose of chylomicron cholesterol administered. Relative rates of hepatic cholesterol synthesis and esterification were measured in isolated hepatocytes. Although hepatic cholesterogenesis increased 300% in diosgenin-fed animals, the contribution of newly synthesized cholesterol to total biliary cholesterol output was only 19 +/- 9%, compared with 12 +/- 6% in control and 15 +/- 5% in diosgenin-fed and EE-injected rats. The rate of oleate incorporation into hepatocytic cholesterol esters was 30% inhibited in diosgenin-fed rats. When EE was injected into these animals, the rate of cholesterol esterification increased to almost 300%. To investigate further the interrelationship between hepatic cholesterol esterification and biliary cholesterol output, we studied 21 diosgenin-fed rats. Six of them received in addition EE and 10 received chylomicron cholesterol. The relationships between biliary cholesterol output as a function of both microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity and hepatic cholesterol ester concentration were significantly correlated in a reciprocal manner. From these

  5. An alternative oxidase monoclonal antibody recognises a highly conserved sequence among alternative oxidase subunits.

    PubMed

    Finnegan, P M; Wooding, A R; Day, D A

    1999-03-19

    The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.

  6. Physiological breeding.

    PubMed

    Reynolds, Matthew; Langridge, Peter

    2016-06-01

    Physiological breeding crosses parents with different complex but complementary traits to achieve cumulative gene action for yield, while selecting progeny using remote sensing, possibly in combination with genomic selection. Physiological approaches have already demonstrated significant genetic gains in Australia and several developing countries of the International Wheat Improvement Network. The techniques involved (see Graphical Abstract) also provide platforms for research and refinement of breeding methodologies. Recent examples of these include screening genetic resources for novel expression of Calvin cycle enzymes, identification of common genetic bases for heat and drought adaptation, and genetic dissection of trade-offs among yield components. Such information, combined with results from physiological crosses designed to test novel trait combinations, lead to more precise breeding strategies, and feed models of genotype-by-environment interaction to help build new plant types and experimental environments for future climates. PMID:27161822

  7. Regulatory Physiology

    NASA Technical Reports Server (NTRS)

    Lane, Helen W.; Whitson, Peggy A.; Putcha, Lakshmi; Baker, Ellen; Smith, Scott M.; Stewart, Karen; Gretebeck, Randall; Nimmagudda, R. R.; Schoeller, Dale A.; Davis-Street, Janis

    1999-01-01

    As noted elsewhere in this report, a central goal of the Extended Duration Orbiter Medical Project (EDOMP) was to ensure that cardiovascular and muscle function were adequate to perform an emergency egress after 16 days of spaceflight. The goals of the Regulatory Physiology component of the EDOMP were to identify and subsequently ameliorate those biochemical and nutritional factors that deplete physiological reserves or increase risk for disease, and to facilitate the development of effective muscle, exercise, and cardiovascular countermeasures. The component investigations designed to meet these goals focused on biochemical and physiological aspects of nutrition and metabolism, the risk of renal (kidney) stone formation, gastrointestinal function, and sleep in space. Investigations involved both ground-based protocols to validate proposed methods and flight studies to test those methods. Two hardware tests were also completed.

  8. Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

    PubMed

    Finnegan, Patrick M; Umbach, Ann L; Wilce, Jackie A

    2003-12-18

    The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

  9. Regulation of NADPH oxidases in skeletal muscle.

    PubMed

    Ferreira, Leonardo F; Laitano, Orlando

    2016-09-01

    The only known function of NAD(P)H oxidases is to produce reactive oxygen species (ROS). Skeletal muscles express three isoforms of NAD(P)H oxidases (Nox1, Nox2, and Nox4) that have been identified as critical modulators of redox homeostasis. Nox2 acts as the main source of skeletal muscle ROS during contractions, participates in insulin signaling and glucose transport, and mediates the myocyte response to osmotic stress. Nox2 and Nox4 contribute to skeletal muscle abnormalities elicited by angiotensin II, muscular dystrophy, heart failure, and high fat diet. Our review addresses the expression and regulation of NAD(P)H oxidases with emphasis on aspects that are relevant to skeletal muscle. We also summarize: i) the most widely used NAD(P)H oxidases activity assays and inhibitors, and ii) studies that have defined Nox enzymes as protagonists of skeletal muscle redox homeostasis in a variety of health and disease conditions. PMID:27184955

  10. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  11. Xanthine Oxidase and Cardiovascular Risk in Obese Children

    PubMed Central

    Tam, Harrison K.; Kelly, Aaron S.; Metzig, Andrea M.; Steinberger, Julia

    2014-01-01

    Abstract Background: Pathological mechanisms of how childhood obesity leads to increased risk of cardiovascular disease (CVD) are not fully characterized. Oxidative-stress–related enzymes, such as xanthine oxidase (XO), have been linked to obesity, endothelial dysfunction, and CVD in adults, but little is known about this pathway in children. The aim of this study was to determine whether differential XO activity is associated with endothelial dysfunction, CVD risk factors, or cytokine levels. Methods: Fasting plasma samples were obtained from obese (BMI ≥95th percentile; n=20) and age- and gender-matched healthy weight (BMI >5th and <85th percentile; n=22) children and adolescents (mean age, 12±3 years) to quantify XO activity. In addition, fasting cholesterol, insulin, glucose, blood pressure, endothelial function, and cytokine levels were assessed. Results: We observed a 3.8-fold increase in plasma XO activity in obese, compared to healthy weight, children (118±21 vs. 31±9 nU/mg of protein; p<0.001). Plasma XO activity was correlated with BMI z-score (r=0.41), waist circumference (r=0.41), high-density lipoprotein cholesterol (r=−0.32), oxidized low-density lipoprotein (r=0.57), adiponectin (r=−0.53), and monocyte chemotactic protein-1 (r=−0.59). Conclusion: XO activity is highly elevated in obese children and correlates with CVD risk factors, suggesting that XO may play a role in increasing cardiovascular risk early in life in the context of obesity. PMID:24568669

  12. Reproductive physiology

    USGS Publications Warehouse

    Gee, G.F.; Russman, S.E.; Ellis, David H.; Gee, George F.; Mirande, Claire M.

    1996-01-01

    Conclusions: Although the general pattern of avian physiology applies to cranes, we have identified many physiological mechanisms (e.g., effects of disturbance) that need further study. Studies with cranes are expensive compared to those done with domestic fowl because of the crane's larger size, low reproductive rate, and delayed sexual maturity. To summarize, the crane reproductive system is composed of physiological and anatomical elements whose function is controlled by an integrated neural-endocrine system. Males generally produce semen at a younger age than when females lay eggs. Eggs are laid in clutches of two (1 to 3), and females will lay additional clutches if the preceding clutches are removed. Both sexes build nests and incubate the eggs. Molt begins during incubation and body molt may be completed annually in breeding pairs. However, remiges are replaced sequentially over 2 to 3 years, or abruptly every 2 to 3 years in other species. Most immature birds replace their juvenal remiges over a 2 to 3 year period. Stress interferes with reproduction in cranes by reducing egg production or terminating the reproductive effort. In other birds, stress elevates corticosterone levels and decreases LHRH release. We know little about the physiological response of cranes to stress.

  13. Reduction of blood serum cholesterol

    NASA Technical Reports Server (NTRS)

    Winitz, M. (Inventor)

    1974-01-01

    By feeding a human subject as the sole source of sustenance a defined diet wherein the carbohydrate consists substantially entirely of glucose, maltose or a polysaccharide of glucose, the blood serum cholesterol level of the human subject is substantially reduced. If 25 percent of the carbohydrate is subsequently supplied in the form of sucrose, an immediate increase from the reduced level is observed. The remainder of the defined diet normally includes a source of amino acids, such as protein or a protein hydrolysate, vitamins, minerals and a source of essential fatty acid.

  14. Azide inhibition of urate oxidase

    PubMed Central

    Gabison, Laure; Colloc’h, Nathalie; Prangé, Thierry

    2014-01-01

    The inhibition of urate oxidase (UOX) by azide was investigated by X-ray diffraction techniques and compared with cyanide inhibition. Two well characterized sites for reagents are present in the enzyme: the dioxygen site and the substrate-binding site. To examine the selectivity of these sites towards azide inhibition, several crystallization conditions were developed. UOX was co-crystallized with azide (N3) in the presence or absence of either uric acid (UA, the natural substrate) or 8-azaxanthine (8AZA, a competitive inhibitor). In a second set of experiments, previously grown orthorhombic crystals of the UOX–UA or UOX–8AZA complexes were soaked in sodium azide solutions. In a third set of experiments, orthorhombic crystals of UOX with the exchangeable ligand 8-nitroxanthine (8NXN) were soaked in a solution containing uric acid and azide simultaneously (competitive soaking). In all assays, the soaking periods were either short (a few hours) or long (one or two months). These different experimental conditions showed that one or other of the sites, or the two sites together, could be inhibited. This also demonstrated that azide not only competes with dioxygen as cyanide does but also competes with the substrate for its enzymatic site. A model in agreement with experimental data would be an azide in equilibrium between two sites, kinetically in favour of the dioxygen site and thermodynamically in favour of the substrate-binding site. PMID:25005084

  15. Heme/copper terminal oxidases

    SciTech Connect

    Ferguson-Miller, S.; Babcock, G.T.

    1996-11-01

    Spatially well-organized electron-transfer reactions in a series of membrane-bound redox proteins form the basis for energy conservation in both photosynthesis and respiration. The membrane-bound nature of the electron-transfer processes is critical, as the free energy made available in exergonic redox chemistry is used to generate transmembrane proton concentration and electrostatic potential gradients. These gradients are subsequently used to drive ATP formation, which provides the immediate energy source for constructive cellular processes. The terminal heme/copper oxidases in respiratory electron-transfer chains illustrate a number of the thermodynamic and structural principles that have driven the development of respiration. This class of enzyme reduces dioxygen to water, thus clearing the respiratory system of low-energy electrons so that sustained electron transfer and free-energy transduction can occur. By using dioxygen as the oxidizing substrate, free-energy production per electron through the chain is substantial, owing to the high reduction potential of O{sub 2} (0.815 V at pH 7). 122 refs.

  16. A Broad Distribution of the Alternative Oxidase in Microsporidian Parasites

    PubMed Central

    Williams, Bryony A. P.; Elliot, Catherine; Burri, Lena; Kido, Yasutoshi; Kita, Kiyoshi; Moore, Anthony L.; Keeling, Patrick J.

    2010-01-01

    Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes. PMID:20169184

  17. Fabrication and characterization of junctionless carbon nanotube field effect transistor for cholesterol detection

    SciTech Connect

    Barik, Md. Abdul Dutta, Jiten Ch.

    2014-08-04

    We have reported fabrication and characterization of polyaniline (PANI)/zinc oxide (ZnO) membrane-based junctionless carbon nanotube field effect transistor deposited on indium tin oxide glass plate for the detection of cholesterol (0.5–22.2 mM). Cholesterol oxidase (ChOx) has been immobilized on the PANI/ZnO membrane by physical adsorption technique. Electrical response has been recorded using digital multimeter (Agilent 3458A) in the presence of phosphate buffer saline of 50 mM, pH 7.0, and 0.9% NaCl contained in a glass pot. The results of response studies for cholesterol reveal linearity as 0.5–16.6 mM and improved sensitivity of 60 mV/decade in good agreement with Nernstian limit ∼59.2 mV/decade. The life time of this sensor has been found up to 5 months and response time of 1 s. The limit of detection with regression coefficient (r) ∼ 0.998 and Michaelis-Menten constant (K{sub m}) were found to be ∼0.25 and 1.4 mM, respectively, indicating high affinity of ChOx to cholesterol. The results obtained in this work show negligible interference with glucose and urea.

  18. Impurity-induced peroxidase mimicry of nanoclay and its potential for the spectrophotometric determination of cholesterol.

    PubMed

    Aneesh, K; Vusa, Chiranjeevi Srinivasa Rao; Berchmans, Sheela

    2016-09-01

    A green version of the "Fe" impurity-induced peroxidase mimicry exhibited by simple and cheap substrate "nanoclay (NC)" along with the highly sensitive amperometric and spectrophotometric determination of cholesterol is demonstrated. The "Fe" impurity can act as the catalyst center for hydrogen peroxide reduction similar to the horseradish peroxidase (HRP)-catalyzed reaction. The Michaelis-Menten constant for the NC-catalyzed reaction is found to be lower than that of the HRP-catalyzed reaction indicating high affinity for the substrate. The NC-modulated peroxidase-like catalytic activity originates from the electron transfer between the reducing substrate in the catalyst center and H2O2 with the intermediate generation of hydroxyl radicals. The peroxidase mimicry is successfully applied for the low-potential electrochemical detection of H2O2 (linear detection range 1.96-10.71 mM, R (2) = 0.97). The H2O2 sensing platform is further modified with cholesterol oxidase (CHOx) for the spectrophotometric (linear detection range 50-244 μM, R (2) = 0.99) and amperometric detection of cholesterol (linear detection range 0.099-1.73 mM, R (2) = 0.998). Graphical abstract Peroxidase mimicry of nanoclay for the determination of cholesterol. PMID:27392749

  19. Development of the layer-by-layer biosensor using graphene films: application for cholesterol determination

    NASA Astrophysics Data System (ADS)

    Binh Nguyen, Hai; Chuc Nguyen, Van; Nguyen, Van Tu; Doan Le, Huu; Quynh Nguyen, Van; Thanh Tam Ngo, Thi; Phuc Do, Quan; Nghia Nguyen, Xuan; Phan, Ngoc Minh; Tran, Dai Lam

    2013-03-01

    The preparation and characterization of graphene films for cholesterol determination are described. The graphene films were synthesized by thermal chemical vapor deposition (CVD) method. Methane gas (CH4) and copper tape were used as carbon source and catalyst in the graphene growth process, respectively. The intergrated array was fabricated by using micro-electro-mechanical systems (MEMS) technology in which Fe3O4-doped polyaniline (PANi) film was electropolymerized on Pt/Gr electrodes. The properties of the Pt/Gr/PANi/Fe3O4 films were investigated by field-emission scanning electron microscopy (FE-SEM), Raman spectroscopy and electrochemical techniques. Cholesterol oxidase (ChOx) has been immobilized onto the working electrode with glutaraldehyde agent. The cholesterol electrochemical biosensor shows high sensitivity (74 μA mM-1 cm-2) and fast response time (<5 s). A linear calibration plot was obtained in the wide cholesterol concentration range from 2 to 20 mM and correlation coefficient square (R2) of 0.9986. This new layer-by-layer biosensor based on graphene films promises many practical applications.

  20. Novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite.

    PubMed

    Ruecha, Nipapan; Rangkupan, Ratthapol; Rodthongkum, Nadnudda; Chailapakul, Orawon

    2014-02-15

    A novel nanocomposite of graphene (G), polyvinylpyrrolidone (PVP) and polyaniline (PANI) has been successfully prepared and used for the modification of paper-based biosensors via electrospraying. The droplet-like nanostructures of G/PVP/PANI-modified electrodes are obtained with an average size of 160 ± 1.02 nm. Interestingly, the presence of small amount of PVP (2 mg mL(-1)) in the nanocomposites can substantially improve the dispersibility of G and increase the electrochemical conductivity of electrodes, leading to enhanced sensitivity of the biosensor. The well-defined cyclic voltammogram of standard ferri/ferrocyanide is achieved on a G/PVP/PANI-modified electrode with a 3-fold increase in the current signal compared to an unmodified electrode. This modified electrode also exhibits excellent electrocatalytic activity towards the oxidation of hydrogen peroxide (H2O2). Furthermore, cholesterol oxidase (ChOx) is attached to G/PVP/PANI-modified electrode for the amperometric determination of cholesterol. Under optimum conditions, a linear range of 50 μM to 10mM is achieved and the limit of detection is found to be 1 μM for cholesterol. Finally, the proposed system can be applied for the determination of cholesterol in a complex biological fluid (i.e. human serum).

  1. Electrochemistry of cholesterol biosensor based on a novel Pt-Pd bimetallic nanoparticle decorated graphene catalyst.

    PubMed

    Cao, Shurui; Zhang, Lei; Chai, Yaqin; Yuan, Ruo

    2013-05-15

    A new electrochemical biosensor with enhanced sensitivity was developed for detection of cholesterol by using platinum-palladium-chitosan-graphene hybrid nanocomposites (PtPd-CS-GS) functionalized glassy carbon electrode (GCE). An electrodeposition method was applied to form PtPd nanoparticles-doped chitosan-graphene hybrid nanocomposites (PtPd-CS-GS), which were characterized by scanning electron microscopy (SEM) and electrochemical methods. The presence of the PtPd-CS-GS nanocomposites not only accelerated direct electron transfer from the redox enzyme to the electrode surface, but also enhanced the immobilized amount of cholesterol oxidase (ChOx). Under optimal conditions, the fabricated biosensor exhibited wide linear ranges of responses to cholesterol in the concentration ranges of 2.2×10(-6) to 5.2×10(-4)M, the limit of detection was 0.75 μM (S/N=3). The response time was less than 7s and the Michaelis-Menten constant (Km(app)) was found as 0.11 mM. In addition, the biosensor also exhibited excellent reproducibility and stability. Along with these attractive features, the biosensor also displayed very high specificity to cholesterol with complete elimination of interference from UA, AA, and glucose. PMID:23618155

  2. Impurity-induced peroxidase mimicry of nanoclay and its potential for the spectrophotometric determination of cholesterol.

    PubMed

    Aneesh, K; Vusa, Chiranjeevi Srinivasa Rao; Berchmans, Sheela

    2016-09-01

    A green version of the "Fe" impurity-induced peroxidase mimicry exhibited by simple and cheap substrate "nanoclay (NC)" along with the highly sensitive amperometric and spectrophotometric determination of cholesterol is demonstrated. The "Fe" impurity can act as the catalyst center for hydrogen peroxide reduction similar to the horseradish peroxidase (HRP)-catalyzed reaction. The Michaelis-Menten constant for the NC-catalyzed reaction is found to be lower than that of the HRP-catalyzed reaction indicating high affinity for the substrate. The NC-modulated peroxidase-like catalytic activity originates from the electron transfer between the reducing substrate in the catalyst center and H2O2 with the intermediate generation of hydroxyl radicals. The peroxidase mimicry is successfully applied for the low-potential electrochemical detection of H2O2 (linear detection range 1.96-10.71 mM, R (2) = 0.97). The H2O2 sensing platform is further modified with cholesterol oxidase (CHOx) for the spectrophotometric (linear detection range 50-244 μM, R (2) = 0.99) and amperometric detection of cholesterol (linear detection range 0.099-1.73 mM, R (2) = 0.998). Graphical abstract Peroxidase mimicry of nanoclay for the determination of cholesterol.

  3. Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts.

    PubMed

    Slotte, J P; Tenhunen, J; Pörn, I

    1990-06-27

    The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.

  4. The Insertion and Transport of Anandamide in Synthetic Lipid Membranes Are Both Cholesterol-Dependent

    PubMed Central

    Di Pasquale, Eric; Chahinian, Henri; Sanchez, Patrick; Fantini, Jacques

    2009-01-01

    Background Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide. Methodology/Principal Findings Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide. Conclusions/Significance Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could

  5. Cholesterol Oxidation in Fish and Fish Products.

    PubMed

    Dantas, Natalie Marinho; Sampaio, Geni Rodrigues; Ferreira, Fernanda Silva; Labre, Tatiana da Silva; Torres, Elizabeth Aparecida Ferraz da Silva; Saldanha, Tatiana

    2015-12-01

    Fish and fish products are important from a nutritional point of view due to the presence of high biological value proteins and the high content of polyunsaturated fatty acids, especially those of the n-3 series, and above all eicosapentaenoic acid and docosahexaenoic acid. However, these important food products also contain significant amounts of cholesterol. Although cholesterol participates in essential functions in the human body, it is unstable, especially in the presence of light, oxygen, radiation, and high temperatures that can cause the formation of cholesterol oxidation products or cholesterol oxides, which are prejudicial to human health. Fish processing involves high and low temperatures, as well as other methods for microbiological control, which increases shelf life and consequently added value; however, such processes favor the formation of cholesterol oxidation products. This review brings together data on the formation of cholesterol oxides during the preparation and processing of fish into food products which are recognized and recommended for their nutritional properties.

  6. Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step.

    PubMed

    Hänsch, Robert; Lang, Christina; Riebeseel, Erik; Lindigkeit, Rainer; Gessler, Arthur; Rennenberg, Heinz; Mendel, Ralf R

    2006-03-10

    Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.

  7. Structure of Cholesterol in Lipid Rafts

    NASA Astrophysics Data System (ADS)

    Toppozini, Laura; Meinhardt, Sebastian; Armstrong, Clare L.; Yamani, Zahra; Kučerka, Norbert; Schmid, Friederike; Rheinstädter, Maikel C.

    2014-11-01

    Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

  8. Macrophage-mediated cholesterol handling in atherosclerosis.

    PubMed

    Chistiakov, Dimitry A; Bobryshev, Yuri V; Orekhov, Alexander N

    2016-01-01

    Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells. PMID:26493158

  9. Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

    SciTech Connect

    Yen, F.T.; Nishida, T. )

    1990-02-01

    In order to efficiently and rapidly label lipoproteins in plasma with ({sup 3}H)cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and ({sup 3}H)cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, ({sup 3}H)cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to ({sup 3}H)cholesterol from an albumin-stabilized emulsion. The distributions of both ({sup 3}H)cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.

  10. Black pepper and piperine reduce cholesterol uptake and enhance translocation of cholesterol transporter proteins.

    PubMed

    Duangjai, Acharaporn; Ingkaninan, Kornkanok; Praputbut, Sakonwun; Limpeanchob, Nanteetip

    2013-04-01

    Black pepper (Piper nigrum L.) lowers blood lipids in vivo and inhibits cholesterol uptake in vitro, and piperine may mediate these effects. To test this, the present study aimed to compare actions of black pepper extract and piperine on (1) cholesterol uptake and efflux in Caco-2 cells, (2) the membrane/cytosol distribution of cholesterol transport proteins in these cells, and (3) the physicochemical properties of cholesterol micelles. Piperine or black pepper extract (containing the same amount of piperine) dose-dependently reduced cholesterol uptake into Caco-2 cells in a similar manner. Both preparations reduced the membrane levels of NPC1L1 and SR-BI proteins but not their overall cellular expression. Micellar cholesterol solubility of lipid micelles was unaffected except by 1 mg/mL concentration of black pepper extract. These data suggest that piperine is the active compound in black pepper and reduces cholesterol uptake by internalizing the cholesterol transporter proteins.

  11. Pigs fed cholesterol neonatally have increased cerebrum cholesterol as young adults.

    PubMed

    Boleman, S L; Graf, T L; Mersmann, H J; Su, D R; Krook, L P; Savell, J W; Park, Y W; Pond, W G

    1998-12-01

    Sixty-eight female neonatal pigs selected for seven (Experiment 1) or eight (Experiment 2) generations for high (HG) or low (LG) plasma cholesterol were used to test the hypothesis that neonatal dietary cholesterol fed during the first 4 or 8 wk of postnatal life increases the cholesterol content of the cerebrum in young adulthood following free access to a high-fat (15%), high-cholesterol (0.5%) diet from 8 to 20 or 24 wk of age. Pigs were removed from their dams at 1 d of age and given free access to a sow-milk replacer diet containing 9.5% coconut fat and 0 or 0.5 % cholesterol. All pigs (except four HG and four LG pigs in Experiment 2, which were deprived of cholesterol throughout the study) were fed the high-fat, high-cholesterol diet from 8 wk to termination at 20 or 24 wk of age. Cerebrum weight and cholesterol concentration were higher in pigs fed cholesterol neonatally than in those deprived of cholesterol neonatally in both experiments, but weight and cholesterol concentration were unaffected by genetic line. Cholesterol concentrations in longissimus and semitendinosus muscles and in subcutaneous fat were unaffected by diet or genetic line. We conclude that dietary cholesterol deprivation during the first 4 to 8 wk of life in piglets is associated with lower cholesterol concentration and total content in the young adult cerebrum than in pigs supplemented with cholesterol in early life. These data support previous observations and suggest the possibility of a metabolic need for neonatal dietary cholesterol in normal brain development. PMID:9868199

  12. Hyperspectral imaging for detection of cholesterol in human skin

    NASA Astrophysics Data System (ADS)

    Milanič, Matija; Bjorgan, Asgeir; Larsson, Marcus; Marraccini, Paolo; Strömberg, Tomas; Randeberg, Lise L.

    2015-03-01

    Hypercholesterolemia is characterized by high levels of cholesterol in the blood and is associated with an increased risk of atherosclerosis and coronary heart disease. Early detection of hypercholesterolemia is necessary to prevent onset and progress of cardiovascular disease. Optical imaging techniques might have a potential for early diagnosis and monitoring of hypercholesterolemia. In this study, hyperspectral imaging was investigated for this application. The main aim of the study was to identify spectral and spatial characteristics that can aid identification of hypercholesterolemia in facial skin. The first part of the study involved a numerical simulation of human skin affected by hypercholesterolemia. A literature survey was performed to identify characteristic morphological and physiological parameters. Realistic models were prepared and Monte Carlo simulations were performed to obtain hyperspectral images. Based on the simulations optimal wavelength regions for differentiation between normal and cholesterol rich skin were identified. Minimum Noise Fraction transformation (MNF) was used for analysis. In the second part of the study, the simulations were verified by a clinical study involving volunteers with elevated and normal levels of cholesterol. The faces of the volunteers were scanned by a hyperspectral camera covering the spectral range between 400 nm and 720 nm, and characteristic spectral features of the affected skin were identified. Processing of the images was done after conversion to reflectance and masking of the images. The identified features were compared to the known cholesterol levels of the subjects. The results of this study demonstrate that hyperspectral imaging of facial skin can be a promising, rapid modality for detection of hypercholesterolemia.

  13. Neonatal dietary cholesterol and alleles of cholesterol 7-alpha hydroxylase affect piglet cerebrum weight, cholesterol concentration, and behavior

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This experiment was designed to test the effect of polymorphism in the cholesterol 7-alpha hydroxylase (CYP7) gene locus, and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG), or low (LG) plasma...

  14. Competition Between Homodimerization and Cholesterol Binding to the C99 Domain of the Amyloid Precursor Protein

    PubMed Central

    Song, Yuanli; Hustedt, Eric J.; Brandon, Suzanne; Sanders, Charles R.

    2013-01-01

    The 99 residue transmembrane C-terminal domain (C99, also known as β-CTF) of the amyloid precursor protein (APP) is the product of β-secretase cleavage of full length APP and the substrate for γ-secretase cleavage. The latter cleavage releases the amyloid-β polypeptides that are closely associated with Alzheimer’s disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes, which center on the transmembrane G700XXXG704XXXG709 glycine zipper motif and the adjacent Gly709. The observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol%) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed Kd for homodimerization (Kd = 0.47 ± 0.15 mol%) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol:C99 complex will be more highly populated than C99 homodimers under most physiological conditions, observations that are of relevance to understanding γ-secretase cleavage of C99. PMID:23865807

  15. Operative treatment of intracranial epidermoid cysts and cholesterol granulomas: report of 21 cases.

    PubMed

    Altschuler, E M; Jungreis, C A; Sekhar, L N; Jannetta, P J; Sheptak, P E

    1990-04-01

    Thirteen patients had operations to remove intracranial epidermoid cysts, and long-term follow-up was obtained. Total or nearly total tumor and capsule removal was accomplished in 7 patients during the initial operation. This group required no additional operations. The other 6 underwent subtotal tumor removal and required multiple operations for symptomatic tumor recurrence. This latter group had a poorer neurological outcome. We conclude that initial total or near-total tumor resection is highly desirable in treating intracranial epidermoid cysts, particularly in physiologically young individuals. Five patients were followed after operations to remove pure cholesterol granulomas of the petroclival bone, and 3 additional patients were followed after operations to remove tumors with combined histopathological features of both an epidermoid cyst and cholesterol granuloma. Four patients with some component of a cholesterol granuloma had concurrent middle ear infections, and 4 did not. Intracranial subtotal excision and drainage of these lesions was the initial operative management in 7 patients, 5 of whom have required multiple operations for symptomatic tumor recurrence. Therefore, we conclude that subtotal excisional procedures for tumors with histopathological features of cholesterol granulomas are not usually successful in establishing long-term cures. Total excision, as recommended for epidermoid cysts, tumors frequently confused with cholesterol granulomas when occupying the petroclival region, may be warranted for these tumors as well. We postulate that when a congenital epidermoid cyst occurs in the petroclival bone, it may incite a local inflammatory reaction, producing lesions which have the histological features of both epidermoid cysts and cholesterol granulomas.

  16. Characteristics and Functional Relevance of Apolipoprotein-A1 and Cholesterol Binding in Mammary Gland Tissues and Epithelial Cells

    PubMed Central

    Ontsouka, Edgar Corneille; Huang, Xiao; Stieger, Bruno; Albrecht, Christiane

    2013-01-01

    Cholesterol in milk is derived from the circulating blood through a complex transport process involving the mammary alveolar epithelium. Details of the mechanisms involved in this transfer are unclear. Apolipoprotein-AI (apoA-I) is an acceptor of cellular cholesterol effluxed by the ATP-binding cassette (ABC) transporter A1 (ABCA1). We aimed to 1) determine the binding characteristics of 125I-apoA-I and 3H-cholesterol to enriched plasma membrane vesicles (EPM) isolated from lactating and non-lactating bovine mammary glands (MG), 2) optimize the components of an in vitro model describing cellular 3H-cholesterol efflux in primary bovine mammary epithelial cells (MeBo), and 3) assess the vectorial cholesterol transport in MeBo using Transwell® plates. The amounts of isolated EPM and the maximal binding capacity of 125I-apoA-I to EPM differed depending on the MG’s physiological state, while the kinetics of 3H-cholesterol and 125I-apoA-I binding were similar. 3H-cholesterol incorporated maximally to EPM after 25±9 min. The time to achieve the half-maximum binding of 125I-apoA-I at equilibrium was 3.3±0.6 min. The dissociation constant (KD) of 125I-apoA-I ranged between 40–74 nmol/L. Cholesterol loading to EPM increased both cholesterol content and 125I-apoA-I binding. The ABCA1 inhibitor Probucol displaced 125I-apoA-I binding to EPM and reduced 3H-cholesterol efflux in MeBo. Time-dependent 3H-cholesterol uptake and efflux showed inverse patterns. The defined binding characteristics of cholesterol and apoA-I served to establish an efficient and significantly shorter cholesterol efflux protocol that had been used in MeBo. The application of this protocol in Transwell® plates with the upper chamber mimicking the apical (milk-facing) and the bottom chamber corresponding to the basolateral (blood-facing) side of cells showed that the degree of 3H-cholesterol efflux in MeBo differed significantly between the apical and basolateral aspects. Our findings support the

  17. NADPH oxidases: new actors in thyroid cancer?

    PubMed

    Ameziane-El-Hassani, Rabii; Schlumberger, Martin; Dupuy, Corinne

    2016-08-01

    Hydrogen peroxide (H2O2) is a crucial substrate for thyroid peroxidase, a key enzyme involved in thyroid hormone synthesis. However, as a potent oxidant, H2O2 might also be responsible for the high level of oxidative DNA damage observed in thyroid tissues, such as DNA base lesions and strand breakages, which promote chromosomal instability and contribute to the development of tumours. Although the role of H2O2 in thyroid hormone synthesis is well established, its precise mechanisms of action in pathological processes are still under investigation. The NADPH oxidase/dual oxidase family are the only oxidoreductases whose primary function is to produce reactive oxygen species. As such, the function and expression of these enzymes are tightly regulated. Thyrocytes express dual oxidase 2, which produces most of the H2O2 for thyroid hormone synthesis. Thyrocytes also express dual oxidase 1 and NADPH oxidase 4, but the roles of these enzymes are still unknown. Here, we review the structure, expression, localization and function of these enzymes. We focus on their potential role in thyroid cancer, which is characterized by increased expression of these enzymes. PMID:27174022

  18. Xanthine oxidase inhibitory activity of alkyl gallates.

    PubMed

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Kubo, Isao

    2006-08-01

    A series (C1-C12) of alkyl gallates was examined for their effects on the activity of xanthine oxidase. Octyl (C8), decyl (C10), and dodecyl (C12) gallates competitively inhibited uric acid formation generated by xanthine oxidase, and the inhibition increased upon increasing the alkyl chain length. Interestingly, neither menthyl nor bornyl gallates inhibited uric acid formation. These data indicate that the hydrophobic alkyl portion is associated with the xanthine-binding site in the Mo-binding domain. It is likely that the linear alkyl portion interacts with the hydrophobic domain close to the binding site, and the hydrophobic interaction is crucial to inhibit the xanthine oxidase reaction. On the other hand, all of gallic acid and its esters equally suppress superoxide anion generation catalyzed by xanthine oxidase at low concentration. The suppression is not due to scavenging activity of these gallates but due to reduction of xanthine oxidase by these gallates. The reduced enzyme catalyzes the reaction to generate hydrogen peroxide and uric acid.

  19. Mitochondrial targeting of human protoporphyrinogen oxidase.

    PubMed

    Davids, Lester M; Corrigall, Anne V; Meissner, Peter N

    2006-05-01

    Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP-causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight protoporphyrinogen oxidase-GFP chimeric fusion proteins created, N-terminal residues 1-17 were found to be the minimal protoporphyrinogen oxidase sequence required for efficient mitochondrial targeting. Removal of this N-terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P-GFP construct abolished mitochondrial targeting, presumably through disruption of the protoporphyrinogen oxidase alpha-helix. Based on our results we propose a mechanism for protoporphyrinogen oxidase targeting to the mitochondrion.

  20. Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: evidence for resolution from a polyphenol oxidase isozyme.

    PubMed

    Ignatov, A; Neuman, M C; Barg, R; Krueger, R J; Coscia, C J

    1997-11-15

    In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria canadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower Mr bands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower Mr bands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases have Mr values of 77 and 70 kDa, respectively, and the two enzymes are different entities.

  1. Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes

    PubMed Central

    Osuda, Yukiho; Shinzawa-Itoh, Kyoko; Tani, Kazutoshi; Maeda, Shintaro; Yoshikawa, Shinya; Tsukihara, Tomitake; Gerle, Christoph

    2016-01-01

    Mitochondrial cytochrome c oxidase utilizes electrons provided by cytochrome c for the active vectorial transport of protons across the inner mitochondrial membrane through the reduction of molecular oxygen to water. Direct structural evidence on the transient cytochrome c oxidase–cytochrome c complex thus far, however, remains elusive and its physiological relevant oligomeric form is unclear. Here, we report on the 2D crystallization of monomeric bovine cytochrome c oxidase with tightly bound cytochrome c at a molar ratio of 1:1 in reconstituted lipid membranes at the basic pH of 8.5 and low ionic strength. PMID:26754561

  2. [Aviation physiology].

    PubMed

    Frank, P W

    1999-10-01

    Aviation physiology should be known at least in parts by the physicians advising air travellers. Due to reducing atmospheric pressure at altitude gas volume in body cavities expands (Boyle's law). This might not be a problem during ascend since air can disappear easily through natural ways. However, air must return to body cavities during descend and a person with a cold may suffer from painful barotitis. Hypoxia is mostly due to a reduced pO2 in high altitude (Daltons's Law). This may be prevented by an aircraft cabin or supplemented oxygen. Decompression sickness is very rare in aviation but divers should comply to a dive free interval before flying. PMID:10568247

  3. Total cholesterol, low density lipoprotein cholesterol, and high density lipoprotein cholesterol and coronary heart disease in Scotland.

    PubMed Central

    Hargreaves, A D; Logan, R L; Thomson, M; Elton, R A; Oliver, M F; Riemersma, R A

    1991-01-01

    OBJECTIVE--To investigate long term changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations and in measures of other risk factors for coronary heart disease and to assess their importance for the development of coronary heart disease in Scottish men. DESIGN--Longitudinal study entailing follow up in 1988-9 of men investigated during a study in 1976. SETTING--Edinburgh, Scotland. SUBJECTS--107 men from Edinburgh who had taken part in a comparative study of risk factors for heart disease with Swedish men in 1976 when aged 40. INTERVENTION--The men were invited to attend a follow up clinic in 1988-9 for measurement of cholesterol concentrations and other risk factor measurements. Eighty three attended and 24 refused to or could not attend. MAIN OUTCOME MEASURES--Changes in total cholesterol, high density lipoprotein cholesterol, and low density lipoprotein cholesterol concentrations, body weight, weight to height index, prevalence of smoking, and alcohol intake; number of coronary artery disease events. RESULTS--Mean serum total cholesterol concentration increased over the 12 years mainly due to an increase in the low density lipoprotein cholesterol fraction (from 3.53 (SD 0.09) to 4.56 (0.11) mmol/l) despite a reduction in high density lipoprotein cholesterol concentration. Body weight and weight to height index increased. Fewer men smoked more than 15 cigarettes/day in 1988-9 than in 1976. Blood pressure remained stable and fasting triglyceride concentrations did not change. The frequency of corneal arcus doubled. Alcohol consumption decreased significantly. Eleven men developed clinical coronary heart disease. High low density lipoprotein and low high density lipoprotein cholesterol concentrations in 1976, but not total cholesterol concentration, significantly predicted coronary heart disease (p = 0.05). Almost all of the men who developed coronary heart disease were smokers (91% v 53%, p less than

  4. Prosopis farcta beans increase HDL cholesterol and decrease LDL cholesterol in ostriches (Struthio camelus).

    PubMed

    Omidi, Arash; Ansari nik, Hossein; Ghazaghi, Mahmood

    2013-02-01

    Ten blue-neck male ostriches (Struthio camelus) were fed Prosopis farcta beans throughout a 30-day experiment. Blood samples were collected from ostriches on days 0 and 30 to measure levels of high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride, total serum protein, albumin, globulin, cholesterol, calcium, inorganic phosphorus, the activity of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transferase (γ-GT). From days 0 to 30, HDL cholesterol, total protein, and globulins levels increased significantly whereas LDL cholesterol, inorganic phosphorus, and γ-GT activity decreased significantly.

  5. Analysis of cholesterol trafficking with fluorescent probes

    PubMed Central

    Maxfield, Frederick R.; Wüstner, Daniel

    2013-01-01

    Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly. PMID:22325611

  6. Cholesterol granulomas in three meerkats (Suricata suricatta).

    PubMed

    Sladky, K K; Dalldorf, F G; Steinberg, H; Wright, J F; Loomis, M R

    2000-11-01

    Cholesterol granulomas are uncommon pathologic lesions in animals, although they are important intracranial tumors in humans. This report describes cholesterol granulomas associated with multiple organ systems of three captive meerkats. In the most severe case, meerkat No. 1, the pathologic behavior of the cholesterol granuloma was unique in that it appeared to locally invade the cerebrum and calvarium, possibly contributing to neurological deficits observed antemortem. A review of other meerkat necropsies revealed incidental, asymptomatic cholesterol granulomas in organs of two other individuals, meerkat Nos. 2 and 3. Histologically, all lesions were composed of cholesterol clefts admixed with large, foamy macrophages containing hemosiderin, multinucleated giant cells, lymphocytes, plasma cells, and foci of mineralization. Hypercholesterolemia was documented in two of the three meerkats.

  7. Cholesterol in myelin biogenesis and hypomyelinating disorders.

    PubMed

    Saher, Gesine; Stumpf, Sina Kristin

    2015-08-01

    The largest pool of free cholesterol in mammals resides in myelin membranes. Myelin facilitates rapid saltatory impulse propagation by electrical insulation of axons. This function is achieved by ensheathing axons with a tightly compacted stack of membranes. Cholesterol influences myelination at many steps, from the differentiation of myelinating glial cells, over the process of myelin membrane biogenesis, to the functionality of mature myelin. Cholesterol emerged as the only integral myelin component that is essential and rate-limiting for the development of myelin in the central and peripheral nervous system. Moreover, disorders that interfere with sterol synthesis or intracellular trafficking of cholesterol and other lipids cause hypomyelination and neurodegeneration. This review summarizes recent results on the roles of cholesterol in CNS myelin biogenesis in normal development and under different pathological conditions. This article is part of a Special Issue entitled Brain Lipids.

  8. Cholesterol granulomas in three meerkats (Suricata suricatta).

    PubMed

    Sladky, K K; Dalldorf, F G; Steinberg, H; Wright, J F; Loomis, M R

    2000-11-01

    Cholesterol granulomas are uncommon pathologic lesions in animals, although they are important intracranial tumors in humans. This report describes cholesterol granulomas associated with multiple organ systems of three captive meerkats. In the most severe case, meerkat No. 1, the pathologic behavior of the cholesterol granuloma was unique in that it appeared to locally invade the cerebrum and calvarium, possibly contributing to neurological deficits observed antemortem. A review of other meerkat necropsies revealed incidental, asymptomatic cholesterol granulomas in organs of two other individuals, meerkat Nos. 2 and 3. Histologically, all lesions were composed of cholesterol clefts admixed with large, foamy macrophages containing hemosiderin, multinucleated giant cells, lymphocytes, plasma cells, and foci of mineralization. Hypercholesterolemia was documented in two of the three meerkats. PMID:11105964

  9. Xanthine oxidase inhibitors from Brandisia hancei.

    PubMed

    Kong, L D; Wolfender, J L; Cheng, C H; Hostettmann, K; Tan, R X

    1999-12-01

    Xanthine oxidase is a key enzyme associated with the incidence of hyperuricemia-related disorders. Repeated chromatography of the enzyme inhibitory part of the water extract of the twigs and leaves of Brandisia hancei (Scrophulariaceae) gave a flavone luteolin, an iridoid glycoside mussaenoside, two beta-sitosterol glycosides daucosterol and beta-sitosterol gentiobioside, and five phenylethanoids arenarioside, brandioside, acteoside, 2'-O-acetylacteoside and isoacteoside. Luteolin and isoacteoside inhibited the xanthine oxidase (XO, EC 1.2.3.2) with the IC50 values at 7.83 and 45.48 microM, respectively. Isoacteoside was found to be the first phenylethanoid that decreased substantially the formation of uric acid by inhibiting competitively xanthine oxidase (Ki value: 10.08 microM). Furthermore, the study suggested that the caffeoylation of the 6'-hydroxyl group of the phenylethanoids was essential for the enzyme inhibitory action.

  10. ALTERNATIVE OXIDASE: From Gene to Function.

    PubMed

    Vanlerberghe, Greg C.; McIntosh, Lee

    1997-06-01

    Plants, some fungi, and protists contain a cyanide-resistant, alternative mitochondrial respiratory pathway. This pathway branches at the ubiquinone pool and consists of an alternative oxidase encoded by the nuclear gene Aox1. Alternative pathway respiration is only linked to proton translocation at Complex 1 (NADH dehydrogenase). Alternative oxidase expression is influenced by stress stimuli-cold, oxidative stress, pathogen attack-and by factors constricting electron flow through the cytochrome pathway of respiration. Control is exerted at the levels of gene expression and in response to the availability of carbon and reducing potential. Posttranslational control involves reversible covalent modification of the alternative oxidase and activation by specific carbon metabolites. This dynamic system of coarse and fine control may function to balance upstream respiratory carbon metabolism and downstream electron transport when these coupled processes become imbalanced as a result of changes in the supply of, or demand for, carbon, reducing power, and ATP.

  11. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    SciTech Connect

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir; Orr, William C.; Sohal, Rajindar S.

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  12. Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers.

    PubMed

    Marri, Costanza; Frazzoli, Alessandra; Hochkoeppler, Alejandro; Poggi, Valeria

    2003-08-01

    A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5. PMID:12877914

  13. In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria

    PubMed Central

    Dahiya, Tulika; Pundir, C.S.

    2013-01-01

    Background & objectives: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. Methods: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. Results: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B6 deficient fed rats as compared with 30 per cent casein vitamin B6 deficient fed rats and control rats. Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones. PMID:23481063

  14. Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.

    PubMed

    Härmälä, A S; Pörn, M I; Mattjus, P; Slotte, J P

    1994-03-24

    The compound U1866A (3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to inhibit the cellular transfer of low-density lipoprotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from plasma membranes to intracellular membranes. Translocation of plasma membrane cholesterol was induced by degradation of plasma membrane sphingomyelin. The sphingomyelinase-induced activation of the acyl-CoA cholesterol acyl transferase (ACAT) reaction was completely inhibited in a dose-dependent manner by U18666A, both in cultured human skin fibroblasts and baby hamster kidney cells. Half-maximal inhibition (within 60 min) was obtained with 0.5-1 microgram/ml of U18666A. A time-course study indicated that the onset of inhibition was rapid (within 10-15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma membrane cholesterol translocation in sphingomyelinase-treated HSF cells was significantly lowered in the presence of U18666A (at 3 micrograms/ml). The effect of U18666A on cholesterol translocation was also fully reversible when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [3H]cholesterol, the compound U18666A inhibited in a dose-dependent manner the cyclic AMP-stimulated secretion of [3H]steroid hormones. The effects seen with compound U18666A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol translocation or ACAT activation. Since different cell types display sensitivity to U18666A in various intracellular cholesterol transfer processes, they appear to have a common U18666A-sensitive regulatory mechanism.

  15. The Effects of Phellinus linteus Polysaccharide Extracts on Cholesterol Efflux in Oxidized Low-Density Lipoprotein-Loaded THP-1 Macrophages.

    PubMed

    Li, Xiao-hui; Li, Yan; Cheng, Zhao-yun; Cai, Xi-guo; Wang, Hong-min

    2015-06-01

    The removal of excess cellular cholesterol is critical for maintaining cellular cholesterol homeostasis. Phellinus linteus polysaccharide extracts (PLPEs) is an immunomudulatory agent with a molecular weight of 153 kd. Here, we analyzed the effects of PLPEs on cholesterol efflux in oxidized low-density lipoprotein (ox-LDL)-loaded THP-1 (human acute monocytic leukemia cell line) macrophages. Various concentrations of PLPEs (5, 10, 20, and 100 μg/mL) were used to treat cells. Cholesterol efflux analysis was performed to analyze the cholesterol efflux ratio in PLPE-treated cells. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were conducted to assess the expression of target genes. Low dose of PLPEs (5-20 μg/mL) dose dependently enhanced cholesterol efflux to apolipoprotein A-I (ApoA-I), evidenced by promoting the expression of adenosine 5'-triphosphate (ATP)-binding cassette A1, ATP-binding cassette G1, and peroxisome proliferation-activated receptor γ, key regulators for cholesterol efflux. Moreover, GW9662, a potent antagonist of peroxisome proliferation-activated receptor γ, inhibited PLPE (20 μg/mL)-promoted cholesterol efflux to ApoA-I in a dose-dependent fashion. However, high dose of PLPEs (100 μg/mL) inhibited cholesterol efflux to ApoA-I from ox-LDL-loaded THP-1 macrophages, enhanced the production of superoxide anion, decreased mitochondrial membrane potential and ATP levels, and raised nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate oxidase subunits. Thus, these results indicate that low and high doses of PLPEs exhibit opposite effects on cholesterol efflux from ox-LDL-loaded THP-1 cells.

  16. Cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.

    PubMed

    Härmälä, A S; Pörn, M I; Mattjus, P; Slotte, J P

    1994-03-24

    The compound U1866A (3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one) has been shown to inhibit the cellular transfer of low-density lipoprotein-derived cholesterol from lysosomes to plasma membranes (Liscum and Faust (1989) J. Biol. Chem. 264, 11796-806). We have in this study examined the effects of U18666A on cholesterol translocation from plasma membranes to intracellular membranes. Translocation of plasma membrane cholesterol was induced by degradation of plasma membrane sphingomyelin. The sphingomyelinase-induced activation of the acyl-CoA cholesterol acyl transferase (ACAT) reaction was completely inhibited in a dose-dependent manner by U18666A, both in cultured human skin fibroblasts and baby hamster kidney cells. Half-maximal inhibition (within 60 min) was obtained with 0.5-1 microgram/ml of U18666A. A time-course study indicated that the onset of inhibition was rapid (within 10-15 min), and reversible if U18666A was removed from the incubation mixture. Using a cholesterol oxidase assay, we observed that the extent of plasma membrane cholesterol translocation in sphingomyelinase-treated HSF cells was significantly lowered in the presence of U18666A (at 3 micrograms/ml). The effect of U18666A on cholesterol translocation was also fully reversible when the drug was withdrawn. In mouse Leydig tumor cells, labeled to constant specific activity with [3H]cholesterol, the compound U18666A inhibited in a dose-dependent manner the cyclic AMP-stimulated secretion of [3H]steroid hormones. The effects seen with compound U18666A appeared to be specific for this molecule, since another hydrophobic amine, imipramine, did not in our experiments affect cholesterol translocation or ACAT activation. Since different cell types display sensitivity to U18666A in various intracellular cholesterol transfer processes, they appear to have a common U18666A-sensitive regulatory mechanism. PMID:8130265

  17. Cubic Phases in Phosphatidylcholine-Cholesterol Mixtures: Cholesterol as Membrane 'Fusogen'

    SciTech Connect

    Tenchov, Boris G.; MacDonald, Robert C.; Siegel, David P.

    2010-01-18

    X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50nm and 25-30nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the L{alpha}HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial L{sub {alpha}} phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.

  18. Physiological Acoustics

    NASA Astrophysics Data System (ADS)

    Young, Eric D.

    The analysis of physiological sound in the peripheral auditory system solves three important problems. First, sound energy impinging on the head must be captured and presented to the transduction apparatus in the ear as a suitable mechanical signal; second, this mechanical signal needs to be transduced into a neural representation that can be used by the brain; third, the resulting neural representation needs to be analyzed by central neurons to extract information useful to the animal. This chapter provides an overview of some aspects of the first two of these processes. The description is entirely focused on the mammalian auditory system, primarily on human hearing and on the hearing of a few commonly used laboratory animals (mainly rodents and carnivores). Useful summaries of non-mammalian hearing are available [1]. Because of the large size of the literature, review papers are referenced wherever possible.

  19. Cholesterol Asymmetry in Synaptic Plasma Membranes

    PubMed Central

    Wood, W. Gibson; Igbavboa, Urule; Müller, Walter E.; Eckert, Gunter P.

    2010-01-01

    Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: 1) chronic ethanol consumption; 2) statins; 3) aging; and 4) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density-lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, p-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function. PMID:21214553

  20. Perturbed cholesterol homeostasis in aging spinal cord.

    PubMed

    Parkinson, Gemma M; Dayas, Christopher V; Smith, Doug W

    2016-09-01

    The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord function is affected by aging, however, the mechanisms involved are not well-understood. To characterize molecular mechanisms of spinal cord aging, microarray analyses of gene expression were performed on cervical spinal cords of aging rats. Of the metabolic and signaling pathways affected, cholesterol-associated pathways were the most comprehensively altered, including significant downregulation of cholesterol synthesis-related genes and upregulation of cholesterol transport and metabolism genes. Paradoxically, a significant increase in total cholesterol content was observed-likely associated with cholesterol ester accumulation. To investigate potential mechanisms for the perturbed cholesterol homeostasis, we quantified the expression of myelin and neuroinflammation-associated genes and proteins. Although there was minimal change in myelin-related expression, there was an increase in phagocytic microglial and astrogliosis markers, particularly in the white matter. Together, these results suggest that perturbed cholesterol homeostasis, possibly as a result of increased inflammatory activation in spinal cord white matter, may contribute to impaired spinal cord function with aging.

  1. Tau pathology induces intraneuronal cholesterol accumulation.

    PubMed

    Glöckner, Frauke; Ohm, Thomas G

    2014-09-01

    Epidemiologic and experimental data suggest the involvement of cholesterol metabolism in the development and progression of Alzheimer disease and Niemann-Pick type C disease, but not of frontotemporal dementias. In these 3 neurodegenerative diseases, however, protein tau hyperphosphorylation and aggregation into neurofibrillary tangles are observed. To elucidate the relationship between cholesterol and tau, we compared sterol levels of neurons burdened with neurofibrillary tangles with those of their unaffected neighbors using semiquantitative filipin fluorescence microscopy in mice expressing P301L mutant human tau (a well-described model of FTDP-17) and in P301L transgenic mice lacking apolipoprotein E (the major cholesterol transporter in the brain). Cellular unesterified cholesterol was higher in neurons affected by tau pathology irrespective of apolipoprotein E deficiency. This argues for an impact of tau pathology on cellular cholesterol homeostasis. We suggest that there is a bidirectional mode of action: Disturbances in cellular cholesterol metabolism may promote tau pathology, but tau pathology may also alter neuronal cholesterol homeostasis; once it is established, a vicious cycle may promote neurofibrillary tangle formation.

  2. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  3. Cholesterol-sensitive Modulation of Transcytosis

    PubMed Central

    Leyt, Julieta; Melamed-Book, Naomi; Vaerman, Jean-Pierre; Cohen, Shulamit; Weiss, Aryeh M.

    2007-01-01

    Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-β-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia. PMID:17392516

  4. Perturbed cholesterol homeostasis in aging spinal cord.

    PubMed

    Parkinson, Gemma M; Dayas, Christopher V; Smith, Doug W

    2016-09-01

    The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord function is affected by aging, however, the mechanisms involved are not well-understood. To characterize molecular mechanisms of spinal cord aging, microarray analyses of gene expression were performed on cervical spinal cords of aging rats. Of the metabolic and signaling pathways affected, cholesterol-associated pathways were the most comprehensively altered, including significant downregulation of cholesterol synthesis-related genes and upregulation of cholesterol transport and metabolism genes. Paradoxically, a significant increase in total cholesterol content was observed-likely associated with cholesterol ester accumulation. To investigate potential mechanisms for the perturbed cholesterol homeostasis, we quantified the expression of myelin and neuroinflammation-associated genes and proteins. Although there was minimal change in myelin-related expression, there was an increase in phagocytic microglial and astrogliosis markers, particularly in the white matter. Together, these results suggest that perturbed cholesterol homeostasis, possibly as a result of increased inflammatory activation in spinal cord white matter, may contribute to impaired spinal cord function with aging. PMID:27459933

  5. Cholesterol content in European bovine milk fats.

    PubMed

    Precht, D

    2001-02-01

    Data about the cholesterol content in edible fats like bovine milk fat are important for balancing the cholesterol intake with food. A comparison of 3 different cholesterol determination methods showed that with the direct analysis by a 25 m long TAP steel capillary column the same results could be obtained as with a time-consuming saponification standard method including thin-layer chromatographic cleaning and subsequent silylation. On the other hand with a rapid direct method using a short packed column 21% unsaponifiables as e.g. minor sterols or hydrocarbons could be found in the "cholesterol peak". The analysis of 1142 German milk fats led to a mean cholesterol content of 265.6 +/- 20.0 mg/100 g fat (range: 204.4 to 382.5). For 165 milk fats from other 12 EU-countries, a similar mean cholesterol content of 258.5 +/- 19.9 mg/100 g fat (range: 215.0 to 331.6) was detected. Compared with sufficiently fed cows, underfed cows demonstrated an approx. 10.1% lower mean cholesterol content (238.7 +/- 9.7 mg/100 g fat). On the other hand, during the first 7 days post partum, the colostrum showed a significantly higher mean cholesterol content of 327.2 +/- 99.0 mg/100 g fat (n = 15; range: 213.1 to 583.9). Further, with special conditions as feeding of rape-seed the cholesterol content can be significantly lowered by 8-13%. An extraordinary lowering up to 50% can be reached by dry fractionation of milk fat (stearin "hard" fraction).

  6. Tissue storage and control of cholesterol metabolism in man on high cholesterol diets.

    PubMed

    Quintão, E C; Brumer, S; Stechhahn, K

    1977-03-01

    The possibility of accumulation of tissue cholesterol in human beings submitted to high cholesterol feeding was investigated in liver biopsies and through fecal sterol balance studies. Feeding to 10 individuals 3.1 to 3.4 g/day of cholesterol for 3 weeks raised the mean serum level from 293 to 349 mg/100 ml, namely 19%, whereas the liver cholesterol content was 417 mg/100 g of wet weight. In 10 control cases eating 0.1--0.4 g/day of cholesterol serum cholesterol remained stable throughout the experimental period and the liver cholesterol content was 256 mg/100 g. Difference of liver colesterol level between the two groups was 62%. In 7 patients submitted to two periods of balance investigation on a cholesterol-free synthetic formula diet respectively prior to (PI) and after (PIII) eating the high cholesterol solid food from 4 to 15 weeks (PII), fecal steroid excretion in PIII exceeded PI in 3 patients. Such data are a direct evidence for the existence of an efficient system to release acutely stored cholesterol. In one patient bile acid excretion accounted for the difference between PIII and PI. PMID:849375

  7. Tissue storage and control of cholesterol metabolism in man on high cholesterol diets.

    PubMed

    Quintão, E C; Brumer, S; Stechhahn, K

    1977-03-01

    The possibility of accumulation of tissue cholesterol in human beings submitted to high cholesterol feeding was investigated in liver biopsies and through fecal sterol balance studies. Feeding to 10 individuals 3.1 to 3.4 g/day of cholesterol for 3 weeks raised the mean serum level from 293 to 349 mg/100 ml, namely 19%, whereas the liver cholesterol content was 417 mg/100 g of wet weight. In 10 control cases eating 0.1--0.4 g/day of cholesterol serum cholesterol remained stable throughout the experimental period and the liver cholesterol content was 256 mg/100 g. Difference of liver colesterol level between the two groups was 62%. In 7 patients submitted to two periods of balance investigation on a cholesterol-free synthetic formula diet respectively prior to (PI) and after (PIII) eating the high cholesterol solid food from 4 to 15 weeks (PII), fecal steroid excretion in PIII exceeded PI in 3 patients. Such data are a direct evidence for the existence of an efficient system to release acutely stored cholesterol. In one patient bile acid excretion accounted for the difference between PIII and PI.

  8. Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study

    PubMed Central

    Mandal, Pritam; Noutsi, Pakiza; Chaieb, Sahraoui

    2016-01-01

    Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells’ behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low. PMID:27245215

  9. Raising HDL cholesterol in women

    PubMed Central

    Eapen, Danny J; Kalra, Girish L; Rifai, Luay; Eapen, Christina A; Merchant, Nadya; Khan, Bobby V

    2010-01-01

    High-density lipoprotein cholesterol (HDL-C) concentration is essential in the determination of coronary heart disease (CHD) risk in women. This is especially true in the postmenopausal state, where lipid profiles and CHD risk mimic that of age-matched men. Thus, interventions designed to reduce CHD risk by raising HDL-C levels may have particular significance during the transition to menopause. This review discusses HDL-C-raising therapies and the role of HDL in the primary prevention of CHD in women. Lifestyle-based interventions such as dietary change, aerobic exercise regimens, and smoking cessation are initial steps that are effective in raising HDL-C, and available data suggest women respond similarly to men with these interventions. When combined with pharmacotherapy, the effects of these lifestyle alterations are further amplified. Though studies demonstrating gender-specific differences in therapy are limited, niacin continues to be the most effective agent in raising HDL-C levels, especially when used in combination with fibrate or statin therapy. Emerging treatments such as HDL mimetic therapy show much promise in further raising HDL-C levels and improving cardiovascular outcomes. PMID:21072287

  10. Genetic therapies to lower cholesterol.

    PubMed

    Khoo, Bernard

    2015-01-01

    This review surveys the state-of-the-art in genetic therapies for familial hypercholesterolaemia (FH), caused most commonly by mutations in the LDL receptor (LDLR) gene. FH manifests as highly elevated low density lipoprotein (LDL) cholesterol levels and consequently accelerated atherosclerosis. Modern pharmacological therapies for FH are insufficiently efficacious to prevent premature cardiovascular disease, can cause significant adverse effects and can be expensive. Genetic therapies for FH have been mooted since the mid 1990s but gene replacement strategies using viral vectors have so far been unsuccessful. Other strategies involve knocking down the expression of Apolipoprotein B100 (APOB100) and the protease PCSK9 which designates LDLR for degradation. The antisense oligonucleotide mipomersen, which knocks down APOB100, is currently marketed (with restrictions) in the USA, but is not approved in Europe due to its adverse effects. To address this problem, we have devised a novel therapeutic concept, APO-skip, which is based on modulation of APOB splicing, and which has the potential to deliver a cost-effective, efficacious and safe therapy for FH.

  11. Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice*

    PubMed Central

    Hager, Lauren; Li, Lixin; Pun, Henry; Liu, Lu; Hossain, Mohammad A.; Maguire, Graham F.; Naples, Mark; Baker, Chris; Magomedova, Lilia; Tam, Jonathan; Adeli, Khosrow; Cummins, Carolyn L.; Connelly, Philip W.; Ng, Dominic S.

    2012-01-01

    We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr−/−xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr−/−xLcat−/− mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr−/−xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr−/−xLcat−/− mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr−/−xLcat−/− mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr−/−xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr−/−xLcat−/− mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance. PMID:22500017

  12. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease. PMID:19141679

  13. Triglycerides, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol in rats exposed to premium motor spirit fumes

    PubMed Central

    Aberare, Ogbevire L.; Okuonghae, Patrick; Mukoro, Nathaniel; Dirisu, John O.; Osazuwa, Favour; Odigie, Elvis; Omoregie, Richard

    2011-01-01

    Background: Deliberate and regular exposure to premium motor spirit fumes is common and could be a risk factor for liver disease in those who are occupationally exposed. A possible association between premium motor spirit fumes and plasma levels of triglyceride, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol using a rodent model could provide new insights in the pathology of diseases where cellular dysfunction is an established risk factor. Aim: The aim of this study was to evaluate the possible effect of premium motor spirit fumes on lipids and lipoproteins in workers occupationally exposed to premium motor spirit fumes using rodent model. Materials and Methods: Twenty-five Wister albino rats (of both sexes) were used for this study between the 4th of August and 7th of September, 2010. The rats were divided into five groups of five rats each. Group 1 rats were not exposed to premium motor spirit fumes (control group), group 2 rats were exposed for 1 hour daily, group 3 for 3 hours daily, group 4 for 5 hours daily and group 5 for 7 hours daily. The experiment lasted for a period of 4 weeks. Blood samples obtained from all the groups after 4 weeks of exposure were used for the estimation of plasma levels of triglyceride, total cholesterol, high density lipoprotein- cholesterol and low density lipoprotein- cholesterol. Result: Results showed significant increase in means of plasma total cholesterol and low density lipoprotein levels (P<0.05). The mean triglyceride and total body weight were significantly lower (P<0.05) in the exposed group when compared with the unexposed. The plasma level of high density lipoprotein, the ratio of low density lipoprotein to high density lipoprotein and the ratio of total cholesterol to high density lipoprotein did not differ significantly in exposed subjects when compared with the control group. Conclusion: These results showed that frequent exposure to petrol fumes may be highly

  14. Copper-Containing Amine Oxidases and FAD-Dependent Polyamine Oxidases Are Key Players in Plant Tissue Differentiation and Organ Development.

    PubMed

    Tavladoraki, Paraskevi; Cona, Alessandra; Angelini, Riccardo

    2016-01-01

    Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell-wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development. PMID:27446096

  15. Copper-Containing Amine Oxidases and FAD-Dependent Polyamine Oxidases Are Key Players in Plant Tissue Differentiation and Organ Development

    PubMed Central

    Tavladoraki, Paraskevi; Cona, Alessandra; Angelini, Riccardo

    2016-01-01

    Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell-wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell-wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and apoplastic metabolic pathways, in tissue differentiation and organ development. PMID:27446096

  16. A Fluorescence Method to Detect and Quantitate Sterol Esterification by Lecithin: Cholesterol Acyltransferase

    PubMed Central

    Homan, Reynold; Esmaeil, Nadia; Mendelsohn, Laurel; Kato, Gregory J.

    2013-01-01

    We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity. PMID:23851343

  17. A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase.

    PubMed

    Homan, Reynold; Esmaeil, Nadia; Mendelsohn, Laurel; Kato, Gregory J

    2013-10-01

    We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity. PMID:23851343

  18. Nanostructured zinc oxide thin film for application to surface plasmon resonance based cholesterol biosensor

    NASA Astrophysics Data System (ADS)

    Kaur, Gurpreet; Tomar, Monika; Gupta, Vinay

    2015-11-01

    ZnO thin film was deposited on gold coated glass prism by RF sputtering technique in glancing angle deposition (GLAD) configuration. The structural, morphological and optical properties of the deposited film were investigated using X-ray diffraction (XRD), Atomic Force Microscopy (AFM) and Fourier Transform Infrared (FTIR) Spectroscopy. ZnO coated Au prisms (ZnO/Au/prism) were used to excite surface plasmons in Kretschmann configuration at the Au- ZnO interface on a laboratory assembled Surface Plasmon Resonance (SPR) measurement setup. Cholesterol oxidase (ChOx) enzyme was immobilized on the ZnO/Au/prism structure by physical adsorption technique. Polydimethylsiloxane (PDMS) microchannels were fabricated over ChOx/ZnO/Au/prism system and various concentrations of cholesterol were passed over the sensor surface. The concentration of cholesterol was varied from 0.12 to 10.23 mM and the SPR reflectance curves were recorded in both static as well as dynamic modes demonstrating a high sensitivity of 0.36° mM-1.

  19. Space Physiology within an Exercise Physiology Curriculum

    ERIC Educational Resources Information Center

    Carter, Jason R.; West, John B.

    2013-01-01

    Compare and contrast strategies remain common pedagogical practices within physiological education. With the support of an American Physiological Society Teaching Career Enhancement Award, we have developed a junior- or senior-level undergraduate curriculum for exercise physiology that compares and contrasts the physiological adaptations of…

  20. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family

    PubMed Central

    Yin, DeLu (Tyler); Urresti, Saioa; Lafond, Mickael; Johnston, Esther M.; Derikvand, Fatemeh; Ciano, Luisa; Berrin, Jean-Guy; Henrissat, Bernard; Walton, Paul H.; Davies, Gideon J.; Brumer, Harry

    2015-01-01

    Alcohol oxidases, including carbohydrate oxidases, have a long history of research that has generated fundamental biological understanding and biotechnological applications. Despite a long history of study, the galactose 6-oxidase/glyoxal oxidase family of mononuclear copper-radical oxidases, Auxiliary Activity Family 5 (AA5), is currently represented by only very few characterized members. Here we report the recombinant production and detailed structure–function analyses of two homologues from the phytopathogenic fungi Colletotrichum graminicola and C. gloeosporioides, CgrAlcOx and CglAlcOx, respectively, to explore the wider biocatalytic potential in AA5. EPR spectroscopy and crystallographic analysis confirm a common active-site structure vis-à-vis the archetypal galactose 6-oxidase from Fusarium graminearum. Strikingly, however, CgrAlcOx and CglAlcOx are essentially incapable of oxidizing galactose and galactosides, but instead efficiently catalyse the oxidation of diverse aliphatic alcohols. The results highlight the significant potential of prospecting the evolutionary diversity of AA5 to reveal novel enzyme specificities, thereby informing both biology and applications. PMID:26680532

  1. Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation.

    PubMed

    Hoober, K L; Thorpe, C

    1999-03-01

    The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.

  2. NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice

    PubMed Central

    Murray, Thomas V.A.; Dong, Xuebin; Sawyer, Greta J.; Caldwell, Anna; Halket, John; Sherwood, Roy; Quaglia, Alberto; Dew, Tracy; Anilkumar, Narayana; Burr, Simon; Mistry, Rajesh K.; Martin, Daniel; Schröder, Katrin; Brandes, Ralf P.; Hughes, Robin D.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins. PMID:26472193

  3. A Multicopper Oxidase-Related Protein Is Essential for Insect Viability, Longevity and Ovary Development

    PubMed Central

    Peng, Zeyu; Green, Peter G.; Arakane, Yasuyuki; Kanost, Michael R.; Gorman, Maureen J.

    2014-01-01

    Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests. PMID:25330116

  4. Swimming physiology.

    PubMed

    Holmér, I

    1992-05-01

    Swimming takes place in a medium, that presents different gravitational and resistive forces, respiratory conditions and thermal stress compared to air. The energy cost of propulsion in swimming is high, but a considerable reduction occurs at a given velocity as result of regular swim training. In medley swimmers the energy cost is lowest for front crawl, followed by backstroke, butterfly and breast-stroke. Cardiac output is probably not limiting for performance since swimmers easily achieve higher values during running. Maximal heart rate, however, is lowered by approx. 10 beats/min during swimming compared to running. Most likely active muscle mass is smaller and rate of power production lesser in swimming. Local factors, such as peripheral circulation, capillary density, perfusion pressure and metabolic capacity of active muscles, are important determinants of the power production capacity and emphasize the role of swim specific training movements. Improved swimming technique and efficiency are likely to explain much of the continuous progress in performance. Rational principles based on improved understanding of the biomechanics and physiology of swimming should be guidelines for swimmers and coaches in their efforts to explore the limits of human performance. PMID:1642724

  5. A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells.

    PubMed

    Ogawa, Yoshikatsu; Tanaka, Mutsuo

    2016-01-01

    Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin-Darby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane.

  6. Extracellular oxidases of the lignin-degrading fungus Panus tigrinus.

    PubMed

    Cadimaliev, D A; Revin, V V; Atykyan, N A; Samuilov, V D

    2005-06-01

    Two extracellular oxidases (laccases) were isolated from the extracellular fluid of the fungus Panus (Lentinus) tigrinus cultivated in low-nitrogen medium supplemented with birch sawdust. The enzymes were purified by successive chromatography on columns with TEAE-cellulose and DEAE-Toyopearl 650M. Both oxidases catalyze oxidation of pyrocatechol and ABTS. Moreover, oxidase 1 also catalyzes oxidation of guaiacol, o-phenylenediamine, and syringaldazine. The enzymes have identical pH (7.0) and temperature (60-65 degrees C) optimums. Absorption spectra of the oxidases differ from the spectra of typical "blue" laccases and are similar to the spectrum of yellow oxidase. PMID:16038613

  7. Understanding the biology of reactive oxygen species and their link to cancer: NADPH oxidases as novel pharmacological targets.

    PubMed

    Harrison, Ian P; Selemidis, Stavros

    2014-08-01

    Reactive oxygen species (ROS), the cellular products of myriad physiological processes, have long been understood to lead to cellular damage if produced in excess and to be a causative factor in cancer through the oxidation and nitration of various macromolecules. Reactive oxygen species influence various hallmarks of cancer, such as cellular proliferation and angiogenesis, through the promotion of cell signalling pathways intrinsic to these processes and can also regulate the function of key immune cells, such as macrophages and regulatory T cells, which promote angiogenesis in the tumour environment. Herein we emphasize the family of NADPH oxidase enzymes as the most likely source of ROS, which promote angiogenesis and tumourigenesis through signalling pathways within endothelial, immune and tumour cells. In this review we focus on the pharmacological inhibitors of NADPH oxidases and suggest that, compared with traditional anti-oxidants, they are likely to offer better alternatives for suppression of tumour angiogenesis. Despite the emerging enthusiasm towards the use of NADPH oxidase inhibitors for cancer therapy, this field is still in its infancy; in particular, there is a glaring lack of knowledge of the roles of NADPH oxidases in in vivo animal models and in human cancers. Certainly a clearer understanding of the relevant signalling pathways influenced by NADPH oxidases during angiogenesis in cancer is likely to yield novel therapeutic approaches.

  8. [Cholesterol and atherosclerosis. Historical considerations and treatment].

    PubMed

    Zárate, Arturo; Manuel-Apolinar, Leticia; Basurto, Lourdes; De la Chesnaye, Elsa; Saldívar, Iván

    2016-01-01

    Cholesterol is a precursor of steroid hormones and an essential component of the cell membrane, however, altered regulation of the synthesis, absorption and excretion of cholesterol predispose to cardiovascular diseases of atherosclerotic origin. Despite, the recognition of historical events for 200 years, starting with Michel Chevreul naming «cholesterol»; later on, Lobstein coining the term atherosclerosis and Marchand introducing it, Anichkov identifying cholesterol in atheromatous plaque, and Brown and Goldstein discovering LDL receptor; as well as the emerging of different drugs, such as fibrates, statins and cetrapibs this decade, promising to increase HDL and the most recent ezetimibe and anti-PCSK9 to inhibit the degradation of LDL receptor, however morbidity has not been reduced in cardiovascular disease. PMID:26774359

  9. [Cholesterol and atherosclerosis. Historical considerations and treatment].

    PubMed

    Zárate, Arturo; Manuel-Apolinar, Leticia; Basurto, Lourdes; De la Chesnaye, Elsa; Saldívar, Iván

    2016-01-01

    Cholesterol is a precursor of steroid hormones and an essential component of the cell membrane, however, altered regulation of the synthesis, absorption and excretion of cholesterol predispose to cardiovascular diseases of atherosclerotic origin. Despite, the recognition of historical events for 200 years, starting with Michel Chevreul naming «cholesterol»; later on, Lobstein coining the term atherosclerosis and Marchand introducing it, Anichkov identifying cholesterol in atheromatous plaque, and Brown and Goldstein discovering LDL receptor; as well as the emerging of different drugs, such as fibrates, statins and cetrapibs this decade, promising to increase HDL and the most recent ezetimibe and anti-PCSK9 to inhibit the degradation of LDL receptor, however morbidity has not been reduced in cardiovascular disease.

  10. HDL Function, Dysfunction, and Reverse Cholesterol Transport

    PubMed Central

    Fisher, Edward A.; Feig, Jonathan E.; Hewing, Bernd; Hazen, Stanley L.; Smith, Jonathan D.

    2012-01-01

    Although high HDL-cholesterol levels are associated with decreased cardiovascular risk in epidemiological studies, recent genetic and pharmacological findings have raised doubts about the beneficial effects of HDL. Raising HDL levels in animal models by infusion or over expression of apolipoprotein A-I has shown clear vascular improvements, such as delayed atherosclerotic lesion progression and accelerated lesion regression, along with increased reverse cholesterol transport. Inflammation and other factors, such as myeloperoxidase mediated oxidation, can impair HDL production and HDL function, in regard to its reverse cholesterol transport, antioxidant, and anti-inflammatory activities. Thus, tests of HDL function, which have not yet been developed as routine diagnostic assays, may prove useful and be a better predictor of cardiovascular risk than HDL-cholesterol levels. PMID:23152494

  11. Serum cholesterol concentration and coronary heart disease in population with low cholesterol concentrations.

    PubMed Central

    Chen, Z; Peto, R; Collins, R; MacMahon, S; Lu, J; Li, W

    1991-01-01

    OBJECTIVE--To examine the relation between serum cholesterol concentration and mortality (from coronary heart disease and from other causes) below the range of cholesterol values generally seen in Western populations. DESIGN--Prospective observational study based on 8-13 years of follow up of subjects in a population with low cholesterol concentrations. SETTING--Urban Shanghai, China. SUBJECTS--9021 Chinese men and women aged 35-64 at baseline. MAIN OUTCOME MEASURE--Death from coronary heart disease and other causes. RESULTS--The average serum cholesterol concentration was 4.2 mmol/l at baseline examination, and only 43 (7%) of the deaths that occurred during 8-13 years of follow up were attributed to coronary heart disease. There was a strongly positive, and apparently independent, relation between serum cholesterol concentration and death from coronary heart disease (z = 3.47, p less than 0.001), and within the range of usual serum cholesterol concentration studied (3.8-4.7 mmol/l) there was no evidence of any threshold. After appropriate adjustment for the regression dilution bias, a 4 (SD 1)% difference in usual cholesterol concentration was associated with a 21 (SD 6)% (95% confidence interval 9% to 35%) difference in mortality from coronary heart disease. There was no significant relation between serum cholesterol concentration and death from stroke or all types of cancer. The 79 deaths due to liver cancer or other chronic liver disease were inversely related to cholesterol concentration at baseline. CONCLUSION--Blood cholesterol concentration was directly related to mortality from coronary heart disease even in those with what was, by Western standards, a "low" cholesterol concentration. There was no good evidence of an adverse effect of cholesterol on other causes of death. PMID:1888927

  12. Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii*

    PubMed Central

    Bottova, Iveta; Hehl, Adrian B.; Štefanić, Saša; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-01-01

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  13. Host cell P-glycoprotein is essential for cholesterol uptake and replication of Toxoplasma gondii.

    PubMed

    Bottova, Iveta; Hehl, Adrian B; Stefanić, Sasa; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Pieters, Jean; Sonda, Sabrina

    2009-06-26

    P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. PMID:19389707

  14. The response of hepatic transcriptome to dietary cholesterol in Prague hereditary hypercholesterolemic (PHHC) rat.

    PubMed

    Vlachová, M; Heczková, M; Jirsa, M; Poledne, R; Kovář, J

    2014-01-01

    To understand the pathogenesis of hypercholesterolemia in Prague hereditary hypercholesterolemic (PHHC) rat, we analyzed the response of hepatic transcriptome to dietary cholesterol in PHHC and control Wistar rats. Male PHHC and Wistar rats were fed chow (C), 5 % fat (palm kernel oil) (CF) or 1 % cholesterol + 5 % fat (CHOL) diet for three weeks. Hepatic transcriptome was analyzed using Affymetrix GeneChip arrays. No differences were found in the effect of both control diets (C and CF) on lipid metabolism and gene expression of 6500 genes. Therefore, these data were pooled for further analysis. Dietary cholesterol induced accumulation of cholesterol and triacylglycerols in the liver in both strains and hypercholesterolemia in PHHC rats. However, there were no differences in response of hepatic transcriptome to CHOL diet. On the other hand, several genes were found to be differently expressed between both strains independently of the diet. Two of those genes, Apof and Aldh1a7, were studied in more detail, and their role in pathogenesis of hypercholesterolemia in PHHC rats could not been corroborated. In conclusion, the hypercholesterolemia in PHHC rats is due to physiological response of hepatic transcriptome to dietary cholesterol in different genetic background.

  15. Properties of ultrathin cholesterol and phospholipid layers surrounding silicon-carbide nanotube: MD simulations.

    PubMed

    Raczyński, Przemysław; Raczyńska, Violetta; Górny, Krzysztof; Gburski, Zygmunt

    2015-08-15

    Computer simulation technique was used to study the dynamics of cholesterol and POPC phospholipid molecules forming a thin layer on the surface of the carbon and silicon-carbide nanotubes. Each nanotube was surrounded by an ultra-thin film formed by n lipid molecules, where n varies from 15 to 50. All studies were done for five temperatures, including physiological one (T=260, 285, 310, 335 and 360K). The influence of a nanotube on the dynamics of cholesterol or phospholipid molecules in a layer is presented and discussed. The water is ubiquitous in all biological milieus, where the cholesterol or lipids occur. Thus, simulations were performed in a water environment. Moreover, to show different behavior of lipids in systems with water the results were compared with the samples without it. The dynamical and structural observables, such as the mean square displacement, diffusion coefficient, radial distribution function, and activation energy were calculated to qualitatively investigate the behavior of cholesterol and phospholipid molecules in the layers. We observed remarkable differences between the cholesterol dynamics depending whether the ultrathin film surrounds carbon or silicon-carbide nanotube and whether the water environment appeared.

  16. [Cholesterol content in longissimus muscle beef from slaughter cattle in Venezuela].

    PubMed

    Huerta Leidenz, N; Ruíz Ramírez, J L; Arenas Moreno, L; Jerez Timaure, N; Márquez, E; Muñoz, B

    1996-12-01

    An observational study was conducted with 149 cattle, raised under tropical conditions of Venezuela (mostly grassfed), to study the relationships of sex class (62 bulls, 67 steers, 20 heifers), age by dentition (2.5; 3.0; 3.5 and 4.0 yr), physiological maturity (A or B), cattle type (17 Dairy or 132 Zebu type crossbreds), Venezuelan carcass grade (Optima, Excelente, Selecta or Superior), marbling level (four levels from "None" to "Small quantity"), carcass fat cover (four level: "Even", "Uneven", "patch-like" and "Devoid") and subcutaneous fat thickness (SFT) over the ribeye (1 = 0.1-0.2 cm; 2 = 0.3-0.4 cm; 3 = 0.5-0.9 cm and 4 = > 1.0 cm) on cholesterol content (mg/ 100 g wet weight) of longissimus muscle. Cholesterol content, as determined colormetrically, did not vary in response to the differences in sex class, age, maturity level, carcass grade, marbling level or SFT represented in the present survey. However, cattle type affected (P = 0.08) cholesterol content. Least square means analysis showed that dairy type contained 12.2 mg more of cholesterol/100 g of muscle than Zebu type. The overall mean (+/- SD) muscle cholesterol for the kind of cattle sampled herein (66.6 +/- 16 mg/100 g) was not considered to be different from those of cattle fed in other latitudes.

  17. Obesity, cholesterol metabolism, and breast cancer pathogenesis.

    PubMed

    McDonnell, Donald P; Park, Sunghee; Goulet, Matthew T; Jasper, Jeff; Wardell, Suzanne E; Chang, Ching-Yi; Norris, John D; Guyton, John R; Nelson, Erik R

    2014-09-15

    Obesity and altered lipid metabolism are risk factors for breast cancer in pre- and post-menopausal women. These pathologic relationships have been attributed in part to the impact of cholesterol on the biophysical properties of cell membranes and to the influence of these changes on signaling events initiated at the membrane. However, more recent studies have indicated that the oxysterol 27-hydroxycholesterol (27HC), and not cholesterol per se, may be the primary biochemical link between lipid metabolism and cancer. The enzyme responsible for production of 27HC from cholesterol, CYP27A1, is expressed primarily in the liver and in macrophages. In addition, significantly elevated expression of this enzyme within breast tumors has also been observed. It is believed that 27HC, acting through the liver X receptor in macrophages and possibly other cells, is involved in maintaining organismal cholesterol homeostasis. It has also been shown recently that 27HC is an estrogen receptor agonist in breast cancer cells and that it stimulates the growth and metastasis of tumors in several models of breast cancer. These findings provide the rationale for the clinical evaluation of pharmaceutical approaches that interfere with cholesterol/27HC synthesis as a means to mitigate the impact of cholesterol on breast cancer pathogenesis. Cancer Res; 74(18); 4976-82. ©2014 AACR. PMID:25060521

  18. Cholesterol suppresses antimicrobial effect of statins

    PubMed Central

    Haeri, Mohammad Reza; White, Kenneth; Qharebeglou, Mohammad; Ansar, Malek Moein

    2015-01-01

    Objective(s): Isoprenoid biosynthesis is a key metabolic pathway to produce a wide variety of biomolecules such as cholesterol and carotenoids, which target cell membranes. On the other hand, it has been reported that statins known as inhibitors of isoprenoid biosynthesis and cholesterol lowering agents, may have a direct antimicrobial effect on the some bacteria. The exact action of statins in microbial metabolism is not clearly understood. It is possible that statins inhibit synthesis or utilization of some sterol precursor necessary for bacterial membrane integrity. Accordingly, this study was designed in order to examine if statins inhibit the production of a compound, which can be used in the membrane, and whether cholesterol would replace it and rescue bacteria from toxic effects of statins. Materials and Methods: To examine the possibility we assessed antibacterial effect of statins with different classes; lovastatin, simvastatin, and atorvastatin, alone and in combination with cholesterol on two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria using gel diffusion assay. Results: Our results showed that all of the statins except for lovastatin had significant antibacterial property in S. aureus, E. coli, and Enter. faecalis. Surprisingly, cholesterol nullified the antimicrobial action of effective statins in statin-sensitive bacteria. Conclusion: It is concluded that statins may deprive bacteria from a metabolite responsible for membrane stability, which is effectively substituted by cholesterol. PMID:26877857

  19. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    SciTech Connect

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-02-27

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in /sup 3/H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture.

  20. Stereoselective recognition of monolayers of cholesterol, ent-cholesterol, and epicholesterol by an antibody.

    PubMed

    Geva, M; Izhaky, D; Mickus, D E; Rychnovsky, S D; Addadi, L

    2001-04-01

    The interaction between a monoclonal antibody and four distinct monolayers with varying degrees of structural, chemical, and stereochemical similarity were studied and quantified. The antibody, raised and selected against cholesterol monohydrate crystals, interacts with cholesterol monolayers stereospecifically, but not enantiospecifically. Monolayers of ent-cholesterol molecules, which are chemically identical to cholesterol and whose structure is the exact mirror image of the cholesterol monolayer, interact with the antibody to the same extent as the cholesterol monolayers. The affinity of the antibody for both enantiomeric monolayers is extremely high. However, the antibody does not interact with monolayers of epicholesterol, which is an epimer of cholesterol: The hydroxy group in epicholesterol is in the 3alpha position rather than in the 3beta position, imposing a different angle between the hydroxy group and the rigid steroid backbone, and a different packing of the molecules. Monolayers of triacontanol, a long-chain primary aliphatic alcohol, interact with the antibody to a lesser extent than the cholesterol and ent-cholesterol monolayers, presumably due to the structural flexibility of the triacontanol molecule. The lack of chiral discrimination by the antibody is thus correlated to the level at which the chirality is exposed at the surface of the monolayers.

  1. The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake*

    PubMed Central

    Zhang, Jin-Hui; Ge, Liang; Qi, Wei; Zhang, Liqing; Miao, Hong-Hua; Li, Bo-Liang; Yang, Maojun; Song, Bao-Liang

    2011-01-01

    Niemann-Pick C1-like 1 (NPC1L1) is a multitransmembrane protein playing a crucial role in dietary and biliary cholesterol absorption. Cholesterol promotes the formation and endocytosis of NPC1L1-flotillin-cholesterol membrane microdomains, which is an early step in cholesterol uptake. How cholesterol is sensed in this step is unknown. Here, we find that the N-terminal domain (NTD) of NPC1L1 binds cholesterol. Mutation of residue Leu-216 in NPC1L1-NTD eliminates cholesterol binding, decreases the formation of NPC1L1-flotillin-cholesterol membrane microdomains, and prevents NPC1L1-mediated cholesterol uptake in culture cells and mice livers. NPC1L1-NTD specifically binds cholesterol but not plant sterols, which may account for the selective cholesterol absorption in intestine. Furthermore, 25- or 27-hydroxycholesterol competes with cholesterol to bind NPC1L1-NTD and inhibits the cholesterol induced endocytosis of NPC1L1. Together, these results demonstrate that plasma membrane-localized NPC1L1 binds exogenous cholesterol via its NTD, and facilitates the formation of NPC1L1-flotillin-cholesterol membrane microdomains that are then internalized into cells through the clathrin-AP2 pathway. Our study uncovers the mechanism of cholesterol sensing by NPC1L1 and proposes a mechanism for selective cholesterol absorption. PMID:21602275

  2. Genetic Alterations Affecting Cholesterol Metabolism and Human Fertility1

    PubMed Central

    DeAngelis, Anthony M.; Roy-O'Reilly, Meaghan; Rodriguez, Annabelle

    2014-01-01

    ABSTRACT Single nucleotide polymorphisms (SNPs) represent genetic variations among individuals in a population. In medicine, these small variations in the DNA sequence may significantly impact an individual's response to certain drugs or influence the risk of developing certain diseases. In the field of reproductive medicine, a significant amount of research has been devoted to identifying polymorphisms which may impact steroidogenesis and fertility. This review discusses current understanding of the effects of genetic variations in cholesterol metabolic pathways on human fertility that bridge novel linkages between cholesterol metabolism and reproductive health. For example, the role of the low-density lipoprotein receptor (LDLR) in cellular metabolism and human reproduction has been well studied, whereas there is now an emerging body of research on the role of the high-density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI) in human lipid metabolism and female reproduction. Identifying and understanding how polymorphisms in the SCARB1 gene or other genes related to lipid metabolism impact human physiology is essential and will play a major role in the development of personalized medicine for improved diagnosis and treatment of infertility. PMID:25122065

  3. The Structural Basis of Cholesterol Activity in Membranes

    SciTech Connect

    Olsen, Brett N.; Bielska, Agata; Lee, Tiffany; Daily, Michael D.; Covey, Douglas F.; Schlesinger, Paul H.; Baker, Nathan A.; Ory, Daniel S.

    2013-10-15

    Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.

  4. The Structural Basis of Cholesterol Accessibility in Membranes

    PubMed Central

    Olsen, Brett N.; Bielska, Agata A.; Lee, Tiffany; Daily, Michael D.; Covey, Douglas F.; Schlesinger, Paul H.; Baker, Nathan A.; Ory, Daniel S.

    2013-01-01

    Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity. PMID:24138860

  5. Hopanoids as functional analogues of cholesterol in bacterial membranes

    PubMed Central

    Sáenz, James P.; Grosser, Daniel; Bradley, Alexander S.; Lagny, Thibaut J.; Lavrynenko, Oksana; Broda, Martyna; Simons, Kai

    2015-01-01

    The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negative Methylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance. PMID:26351677

  6. Hopanoids as functional analogues of cholesterol in bacterial membranes.

    PubMed

    Sáenz, James P; Grosser, Daniel; Bradley, Alexander S; Lagny, Thibaut J; Lavrynenko, Oksana; Broda, Martyna; Simons, Kai

    2015-09-22

    The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negative Methylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.

  7. Expression of a Streptomyces 3-hydroxysteroid oxidase gene in oilseeds for converting phytosterols to phytostanols.

    PubMed

    Venkatramesh, Mylavarapu; Karunanandaa, Balasulojini; Sun, Bin; Gunter, Catharine A; Boddupalli, Sekhar; Kishore, Ganesh M

    2003-01-01

    Plant sterols and their hydrogenated forms, stanols, have attracted much attention because of their benefits to human health in reducing serum and LDL cholesterol levels, with vegetable oil processing being their major source in several food products currently sold. The predominant forms of plant sterol end products are sitosterol, stigmasterol, campesterol and brassicasterol (in brassica). In this study, 3-hydroxysteroid oxidase from Streptomyces hygroscopicus was utilized to engineer oilseeds from rapeseed (Brassica napus) and soybean (Glycine max), respectively, to modify the relative amounts of specific sterols to stanols. Each of the major phytosterols had its C-5 double bond selectively reduced to the corresponding phytostanol without affecting other functionalities, such as the C-22 double bond of stigmasterol in soybean seed and of brassicasterol in rapeseed. Additionally, several novel phytostanols were obtained that are not produced by chemical hydrogenation of phytosterols normally present in plants.

  8. NADPH oxidases in the arbuscular mycorrhizal symbiosis

    PubMed Central

    Belmondo, Simone; Calcagno, Cristina; Genre, Andrea; Puppo, Alain; Pauly, Nicolas; Lanfranco, Luisa

    2016-01-01

    ABSTRACT Plant NADPH oxidases are the major source of reactive oxygen species (ROS) that plays key roles as both signal and stressor in several plant processes, including defense responses against pathogens. ROS accumulation in root cells during arbuscular mycorrhiza (AM) development has raised the interest in understanding how ROS-mediated defense programs are modulated during the establishment of this mutualistic interaction. We have recently analyzed the expression pattern of 5 NADPH oxidase (also called RBOH) encoding genes in Medicago truncatula, showing that only one of them (MtRbohE) is specifically upregulated in arbuscule-containing cells. In line with this result, RNAi silencing of MtRbohE generated a strong alteration in root colonization, with a significant reduction in the number of arbusculated cells. On this basis, we propose that MtRBOHE-mediated ROS production plays a crucial role in the intracellular accommodation of arbuscules. PMID:27018627

  9. Lysyl oxidase isoforms in gastric cancer.

    PubMed

    Añazco, Carolina; Delgado-López, Fernando; Araya, Paulina; González, Ileana; Morales, Erik; Pérez-Castro, Ramón; Romero, Jacqueline; Rojas, Armando

    2016-09-01

    Gastric cancer (GC) is the fifth most frequent cancer in the world and shows the highest incidence in Latin America and Asia. An increasing amount of evidence demonstrates that lysyl oxidase isoforms, a group of extracellular matrix crosslinking enzymes, should be considered as potential biomarkers and therapeutic targets in GC. In this review, we focus on the expression levels of lysyl oxidase isoforms, its functions and the clinical implications in GC. Finding novel proteins related to the processing of these extracellular matrix enzymes might be helpful in the design of new therapies, which, in combination with classic pharmacology, could be used to delay the progress of this aggressive cancer and offer a wider temporal window for clinical intervention. PMID:27564724

  10. Physiological consequences of everyday psychosocial stress.

    PubMed

    Pollard, T M

    1997-06-01

    A large body of data has been accumulated concerning physiological responses in people exposed to stressors in laboratories. Adrenaline and cortisol have become known as "stress hormones" because, in men, levels of both hormones consistently rise in response to stress in laboratory-based investigations. If chronically repeated, elevation of adrenaline and cortisol is likely to have long-term consequences for health, especially cardiovascular health, partly via the effects of the hormones on blood pressure and serum cholesterol levels. Research on people conducting their everyday lives is necessary to establish whether the same responses are shown on a day to day basis. Such research requires new methodologies and careful data collection. So far, it has been shown that adrenaline and blood pressure do seem to vary in expected ways. Other responses in everyday life, including those of cholesterol, cortisol and the immune system, are less well characterised.

  11. Mitochondrial regulation of macrophage cholesterol homeostasis.

    PubMed

    Graham, Annette

    2015-12-01

    This review explores the relationship between mitochondrial structure and function in the regulation of macrophage cholesterol metabolism and proposes that mitochondrial dysfunction contributes to loss of the elegant homeostatic mechanisms which normally maintain cellular sterol levels within defined limits. Mitochondrial sterol 27-hydroxylase (CYP27A1) can generate oxysterol activators of liver X receptors which heterodimerise with retinoid X receptors, enhancing the transcription of ATP binding cassette transporters (ABCA1, ABCG1, and ABCG4), that can remove excess cholesterol via efflux to apolipoproteins A-1, E, and high density lipoprotein, and inhibit inflammation. The activity of CYP27A1 is regulated by the rate of supply of cholesterol substrate to the inner mitochondrial membrane, mediated by a complex of proteins. The precise identity of this dynamic complex remains controversial, even in steroidogenic tissues, but may include steroidogenic acute regulatory protein and the 18 kDa translocator protein, together with voltage-dependent anion channels, ATPase AAA domain containing protein 3A, and optic atrophy type 1 proteins. Certainly, overexpression of StAR and TSPO proteins can enhance macrophage cholesterol efflux to apoA-I and/or HDL, while perturbations in mitochondrial function, or changes in the expression of mitochondrial fusion proteins, alter the efficiency of cholesterol efflux. Molecules which can sustain or improve mitochondrial function or increase the activity of the protein complex involved in cholesterol transfer may have utility in resolving the problem of dysregulated macrophage cholesterol homeostasis, a condition which may contribute to inflammation, atherosclerosis, nonalcoholic steatohepatitis, osteoblastic bone resorption, and some disorders of the central nervous system.

  12. The absorption of cholesterol and the sterol balance in the Tarahumara Indians of Mexico fed cholesterol-free and high cholesterol diets.

    PubMed

    McMurry, M P; Connor, W E; Lin, D S; Cerqueira, M T; Connor, S L

    1985-06-01

    The Tarahumara Indians of Mexico are habituated to a very low cholesterol, low fat diet and have lifelong low plasma cholesterol concentrations. To study cholesterol metabolism in these unusual people, 8 Tarahumara men were fed sequentially a cholesterol-free diet and then a diet containing 900 mg cholesterol under controlled conditions. The intestinal absorption of cholesterol, fecal steroid excretion and sterol balance were determined. During the high cholesterol diet period, the plasma cholesterol level increased from 113 +/- 8 mg/dl to 147 +/- 11 mg/dl (means +/- SD). Cholesterol biosynthesis decreased from 14.0 +/- 0.7 to 7.1 +/- 1.0 mg/kg/day (means +/- SE). The intestinal absorption of cholesterol was 27.7 +/- 6.7% (means +/- SE) during both dietary periods. Compared to other cultures, Tarahumaras had a reduced ability to absorb dietary cholesterol and higher total sterol turnover primarily because of an increased bile acid output. The total sterol disposition over three weeks of the high cholesterol diet accounted for all the absorbed dietary cholesterol.

  13. Imaging Monoamine Oxidase in the Human Brain

    SciTech Connect

    Fowler, J. S.; Volkow, N. D.; Wang, G-J.; Logan, Jean

    1999-11-10

    Positron emission tomography (PET) studies mapping monoamine oxidase in the human brain have been used to measure the turnover rate for MAO B; to determine the minimum effective dose of a new MAO inhibitor drug lazabemide and to document MAO inhibition by cigarette smoke. These studies illustrate the power of PET and radiotracer chemistry to measure normal biochemical processes and to provide information on the effect of drug exposure on specific molecular targets.

  14. Monoamine oxidase inhibitors from Gentiana lutea.

    PubMed

    Haraguchi, Hiroyuki; Tanaka, Yasumasa; Kabbash, Amal; Fujioka, Toshihiro; Ishizu, Takashi; Yagi, Akira

    2004-08-01

    Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3''linked-(2'-hydroxy-4-O-isoprenylchalcone)-(2'''-hydroxy-4''-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1'-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.

  15. Aspirin Prevention of Cholesterol Gallstone Formation in Prairie Dogs

    NASA Astrophysics Data System (ADS)

    Lee, Sum P.; Carey, Martin C.; Lamont, J. Thomas

    1981-03-01

    When prairie dogs (Cynomys ludovicianus) are fed a diet containing cholesterol, a marked increase in gallbladder mucin secretion parallels the evolution of cholesterol supersaturated bile. Gelation of mucin precedes the precipitation of cholesterol liquid and solid crystals and the development of gallstones. Aspirin given to prairie dogs inhibited mucin hypersecretion and gel accumulation and prevented gallstone formation without influencing the cholesterol content of supersaturated bile. This suggests that gallbladder mucin is a nucleation matrix for cholesterol gallstones.

  16. Aronia melanocarpa (chokeberry) polyphenol-rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice.

    PubMed

    Kim, Bohkyung; Ku, Chai Siah; Pham, Tho X; Park, Youngki; Martin, Derek A; Xie, Liyang; Taheri, Rod; Lee, Jiyoung; Bolling, Bradley W

    2013-05-01

    We hypothesized that a polyphenol-rich chokeberry extract (CBE) would modulate hepatic lipid metabolism and improve antioxidant function in apolipoprotein E knockout (apoE(-/-)) mice. ApoE(-/-) mice were fed diets containing 15% fat with 0.2% cholesterol alone or supplemented with 0.005% or 0.05% CBE for 4 weeks. CBE polyphenol content was determined by the total phenols, 4-dimethylaminocinnamaldehyde, and ultra high-performance liquid chromatography-mass spectrometry methods. The 0.05% CBE diet provided mice with mean daily doses of 1.2 mg gallic acid equivalents of total phenols, 0.19 mg anthocyanins, 0.17 mg phenolic acids, 0.06 mg proanthocyanidins (as catechin-equivalents), and 0.02 mg flavonols. The 0.05% CBE group had 12% less plasma total cholesterol concentrations than the control. Despite the hypocholesterolemic effect of CBE, hepatic mRNA levels of low-density lipoprotein receptor, hydroxyl-3-methylglutaryl coenzyme A reductase and cholesterol 7α-hydroxylase in CBE-fed mice were not significantly different from controls. Dietary CBE did not alter hepatic lipid content or the hepatic expression of genes involved in lipogenesis and fatty acid β-oxidation such as fatty acid synthase, carnitine palmitoyltransferase 1 and acyl-CoA oxidase. Plasma paraoxonase and catalase activities were significantly increased in mice fed 0.05% CBE. Both CBE diets increased hepatic glutathione peroxidase (GPx) activity but the 0.05% CBE group had 24% less proximal intestine GPx activity relative to controls. Thus, dietary CBE lowered total cholesterol and improved plasma and hepatic antioxidant function at nutritionally-relevant doses in apoE(-/-) mice. Furthermore, the CBE cholesterol-lowering mechanism in apoE(-/-) mice was independent of hepatic expression of genes involved in cholesterol metabolism.

  17. Fabrication of a novel dual mode cholesterol biosensor using titanium dioxide nanowire bridged 3D graphene nanostacks.

    PubMed

    Komathi, S; Muthuchamy, N; Lee, K-P; Gopalan, A-I

    2016-10-15

    Herein, we fabricated a novel electrochemical-photoelectrochemical (PEC) dual-mode cholesterol biosensor based on graphene (G) sheets interconnected-graphene embedded titanium nanowires (TiO2(G)-NWs) 3D nanostacks (designated as G/Ti(G) 3DNS) by exploiting the beneficial characteristics of G and TiO2-NWs to achieve good selectivity and high sensitivity for cholesterol detection. The G/Ti(G) 3DNS was fabricated by the reaction between functionalized G and TiO2(G)-NWs. Cholesterol oxidase (ChOx) was subsequently immobilized in to G/Ti(G) 3DNS using chitosan (CS) as the binder and the dual mode G/Ti(G) 3DNS/CS/ChOx biosensor was fabricated. The electro-optical properties of the G/Ti(G) 3DNS/CS/ChOx bioelectrode were characterized by cyclic voltammetry and UV-vis diffuse reflection spectroscopy. The cyclic voltammetry of immobilized ChOx showed a pair of well-defined redox peaks indicating direct electron transfer (DET) of ChOx. The amperometric reduction peak current (at -0.05V) linearly increased with increase in cholesterol concentration. The G/Ti(G) 3DNS/CS/ChOx bioelectrode was selective to cholesterol with a remarkable sensitivity (3.82μA/cm(2)mM) and a lower detection limit (6μM). Also, G/Ti(G) 3DNS/CS/ChOx functioned as photoelectrode and exhibited selective detection of cholesterol under a low bias voltage and light irradiation. Kinetic parameters, reproducibility, repeatability, storage stability and effect of temperature and pH were evaluated. We envisage that G/Ti(G) 3DNS with its prospective characteristics, would be a promising material for wide range of biosensing applications.

  18. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    PubMed

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  19. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): enzymes with multiple sterols as substrates and as activators

    PubMed Central

    Rogers, Maximillian A.; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C.Y.; Chang, Ta-Yuan

    2016-01-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the isooctyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  20. Increased xanthine oxidase in the skin of preeclamptic women.

    PubMed

    Bainbridge, Shannon A; Deng, Jau-Shyong; Roberts, James M

    2009-05-01

    Xanthine oxioreductase is the holoenzyme responsible for terminal purine catabolism. Under conditions of metabolic stress or heightened proinflammatory cytokine production, this enzyme is preferentially in its oxidized form, xanthine oxidase, with catalytic action that generates uric acid and the free radical superoxide. As preeclampsia is characterized by heightened inflammation, oxidative stress, and hyperuricemia, it has been proposed that xanthine oxidase plays a pivotal role in this hypertensive disorder of pregnancy. We sought to determine whether xanthine oxidase protein content was higher in maternal tissue of preeclamptic mothers, compared to healthy pregnant controls, using immunohistochemical analysis of skin biopsies. We further compared xanthine oxidase immunoreactivity in skin biopsies from preeclamptic women and patients with several inflammatory conditions. In preeclamptic women, intense xanthine oxidase immunoreactivity was present within the epidermis. By contrast, only very faint xanthine oxidase staining was observed in skin biopsies from healthy pregnant controls. Further, a role for inflammation in the increase of xanthine oxidase was suggested by similar findings of heightened xanthine oxidase immunoreactivity in the skin biopsies from nonpregnant individuals diagnosed with conditions of systemic inflammation. The finding of increased xanthine oxidase in maternal tissue, most likely as the result of heightened maternal inflammation, suggests maternal xanthine oxidase as a source of free radical and uric acid generation in preeclampsia.

  1. Prevention of cholesterol gallstones by inhibiting hepatic biosynthesis and intestinal absorption of cholesterol

    PubMed Central

    Wang, Helen H; Portincasa, Piero; de Bari, Ornella; Liu, Kristina J; Garruti, Gabriella; Neuschwander-Tetri, Brent A; Wang, David Q.-H

    2013-01-01

    Cholesterol cholelithiasis is a multifactorial disease influenced by a complex interaction of genetic and environmental factors, and represents a failure of biliary cholesterol homeostasis in which the physical-chemical balance of cholesterol solubility in bile is disturbed. The primary pathophysiologic event is persistent hepatic hypersecretion of biliary cholesterol, which has both hepatic and small intestinal components. The majority of the environmental factors are probably related to Western-type dietary habits, including excess cholesterol consumption. Laparoscopic cholecystectomy, one of the most commonly performed surgical procedures in the US, is nowadays a major treatment for gallstones. However, it is invasive and can cause surgical complications, and not all patients with symptomatic gallstones are candidates for surgery. The hydrophilic bile acid, ursodeoxycholic acid (UDCA) has been employed as first-line pharmacological therapy in a subgroup of symptomatic patients with small, radiolucent cholesterol gallstones. Long-term administration of UDCA can promote the dissolution of cholesterol gallstones. However, the optimal use of UDCA is not always achieved in clinical practice because of failure to titrate the dose adequately. Therefore, the development of novel, effective, and noninvasive therapies is crucial for reducing the costs of health care associated with gallstones. In this review, we summarize recent progress in investigating the inhibitory effects of ezetimibe and statins on intestinal absorption and hepatic biosynthesis of cholesterol, respectively, for the treatment of gallstones, as well as in elucidating their molecular mechanisms by which combination therapy could prevent this very common liver disease worldwide. PMID:23419155

  2. Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells.

    PubMed

    Wüstner, Daniel; Lund, Frederik W; Röhrl, Clemens; Stangl, Herbert

    2016-01-01

    Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.

  3. Phase separation of cholesterol and the interaction of ethanol with phosphatidylserine-cholesterol bilayer membranes.

    PubMed

    Bach, D; Borochov, N; Wachtel, E

    2002-02-01

    Thermotropic and structural effects of ethanol on phosphatidylserine (PS) membranes containing up to 0.4 mol fraction cholesterol were investigated by differential scanning calorimetry, X-ray diffraction and fluorescence spectroscopy. It was found that in the presence of cholesterol, 10% (v/v) added ethanol depresses the melting temperature of the phospholipid by approximately 2 degrees C, similar to what was observed in the absence of cholesterol. Below the melting temperature the progressive disordering effect of added cholesterol is weakly enhanced by the presence of ethanol. In the liquid crystalline state, the marked decrease in the thickness of the bilayer which ethanol causes in the absence of cholesterol (Chem. Phys. Lipids 92 (1998) 127), is also observed in its presence. We conclude that, in contrast to what has been observed for zwitterionic phospholipids, high concentrations of cholesterol do not diminish the interaction of ethanol with PS membranes. With addition of 10% (v/v) ethanol, crystalline cholesterol diffraction, an indication of phase separation of the sterol, appears at mol fraction cholesterol 0.34, as compared to 0.3 in the absence of ethanol (Chem. Phys. Lipids 92 (1998) 71).

  4. A diet rich in leafy vegetable fiber improves cholesterol metabolism in high-cholesterol fed rats.

    PubMed

    Ezz El-Arab, A M

    2009-10-01

    In the present study, the hypocholesterolemic effect of leaf vegetable (Jew's mallow) was studied in high-cholesterol fed rats. The animals were fed diets supplemented with cholesterol (0.25%) for 4 weeks. Leaf vegetable diet produced an important hypocholesterolemic action: it led to a significant lowering (p<0.05) of cholesterol in the plasma and liver, as well as of the atherogenic index and a significant increase (p<0.05) in cecal short chain fatty acids, with respect to the control group. Concurrently, total fecal neutral sterols in the excretion increased (p<0.05) and apparent absorption of dietary cholesterol was significantly depressed (-58%). The consumption of leaf vegetable (Jew's mallow) with a hypercholesterolemic diet improved the lipidemic profile and increased excretion of the total cholesterol end-products. PMID:20387744

  5. A diet rich in leafy vegetable fiber improves cholesterol metabolism in high-cholesterol fed rats.

    PubMed

    Ezz El-Arab, A M

    2009-10-01

    In the present study, the hypocholesterolemic effect of leaf vegetable (Jew's mallow) was studied in high-cholesterol fed rats. The animals were fed diets supplemented with cholesterol (0.25%) for 4 weeks. Leaf vegetable diet produced an important hypocholesterolemic action: it led to a significant lowering (p<0.05) of cholesterol in the plasma and liver, as well as of the atherogenic index and a significant increase (p<0.05) in cecal short chain fatty acids, with respect to the control group. Concurrently, total fecal neutral sterols in the excretion increased (p<0.05) and apparent absorption of dietary cholesterol was significantly depressed (-58%). The consumption of leaf vegetable (Jew's mallow) with a hypercholesterolemic diet improved the lipidemic profile and increased excretion of the total cholesterol end-products.

  6. [Trans-intestinal cholesterol excretion (TICE): a new route for cholesterol excretion].

    PubMed

    Blanchard, Claire; Moreau, François; Cariou, Bertrand; Le May, Cédric

    2014-10-01

    The small intestine plays a crucial role in dietary and biliary cholesterol absorption, as well as its lymphatic secretion as chylomicrons (lipoprotein exogenous way). Recently, a new metabolic pathway called TICE (trans-intestinal excretion of cholesterol) that plays a central role in cholesterol metabolism has emerged. TICE is an inducible way, complementary to the hepatobiliary pathway, allowing the elimination of the plasma cholesterol directly into the intestine lumen through the enterocytes. This pathway is poorly characterized but several molecular actors of TICE have been recently identified. Although it is a matter of debate, two independent studies suggest that TICE is involved in the anti-atherogenic reverse cholesterol transport pathway. Thus, TICE is an innovative drug target to reduce -cardiovascular diseases.

  7. Glucagon receptor antagonism induces increased cholesterol absorption.

    PubMed

    Guan, Hong-Ping; Yang, Xiaodong; Lu, Ku; Wang, Sheng-Ping; Castro-Perez, Jose M; Previs, Stephen; Wright, Michael; Shah, Vinit; Herath, Kithsiri; Xie, Dan; Szeto, Daphne; Forrest, Gail; Xiao, Jing Chen; Palyha, Oksana; Sun, Li-Ping; Andryuk, Paula J; Engel, Samuel S; Xiong, Yusheng; Lin, Songnian; Kelley, David E; Erion, Mark D; Davis, Harry R; Wang, Liangsu

    2015-11-01

    Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

  8. Dual roles for cholesterol in mammalian cells.

    PubMed

    Xu, Fang; Rychnovsky, Scott D; Belani, Jitendra D; Hobbs, Helen H; Cohen, Jonathan C; Rawson, Robert B

    2005-10-11

    The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, beta-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in beta-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in beta-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective. PMID:16199524

  9. LDL cholesterol: controversies and future therapeutic directions.

    PubMed

    Ridker, Paul M

    2014-08-16

    Lifelong exposure to raised concentrations of LDL cholesterol increases cardiovascular event rates, and the use of statin therapy as an adjunct to diet, exercise, and smoking cessation has proven highly effective in reducing the population burden associated with hyperlipidaemia. Yet, despite consistent biological, genetic, and epidemiological data, and evidence from randomised trials, there is controversy among national guidelines and clinical practice with regard to LDL cholesterol, its measurement, the usefulness of population-based screening, the net benefit-to-risk ratio for different LDL-lowering drugs, the benefit of treatment targets, and whether aggressive lowering of LDL is safe. Several novel therapies have been introduced for the treatment of people with genetic defects that result in loss of function within the LDL receptor, a major determinant of inherited hyperlipidaemias. Moreover, the usefulness of monoclonal antibodies that extend the LDL-receptor lifecycle (and thus result in substantial lowering of LDL cholesterol below the levels achieved with statins alone) is being assessed in phase 3 trials that will enrol more than 60,000 at-risk patients worldwide. These trials represent an exceptionally rapid translation of genetic observations into clinical practice and will address core questions of how low LDL cholesterol can be safely reduced, whether the mechanism of LDL-cholesterol lowering matters, and whether ever more aggressive lipid-lowering provides a safe, long-term mechanism to prevent atherothrombotic complications.

  10. Plasma cholesterol transport in anhepatic rats.

    PubMed Central

    Quarfordt, S H; Landis, B; Cucchiaro, G; Yamaguchi, Y; Oswald, B

    1992-01-01

    The plasma appearance of newly synthesized cholesterol in anhepatic laboratory diet-fed rats was 10% of the intact rat. In intact rats this cholesterol was mainly ester in lower density lipoproteins, but for anhepatic rats it was virtually only free in high density lipoprotein. Chylomicron cholesterol ester was removed much more slowly from anhepatic than control plasma and returned primarily as free in high density lipoproteins, with the control return 10 times the anhepatic return. Lower density lipoprotein cholesterol ester transfer to an extravascular pool in anhepatic rats was less than 10% of controls. The liver was responsible for 95% of the extravascular lower density lipoprotein ester pool and only 50% of the for high density lipoprotein ester. Despite decreased anhepatic lipoprotein catabolism, the mass of both plasma low and high density lipoproteins progressively decreased indicating an even greater decrease in influx. The anhepatic fractional catabolic rate of apo A1 was similar to controls, but that of apo E was considerably less. Despite the unchanged catabolism of apo A1 and the reduced catabolism of apo E, plasma apo A1 decreased less than apo E after hepatectomy. The anhepatic data confirm the pivotal role of the liver in maintaining plasma low and high density lipoprotein cholesterol concentrations. They suggest that, in addition to its anabolic and catabolic functions, the liver also acts as a reservoir buffering changes in plasma concentration. Images PMID:1569195

  11. Evolution of resistance to phytoene desaturase and protoporphyrinogen oxidase inhibitors--state of knowledge.

    PubMed

    Dayan, Franck E; Owens, Daniel K; Tranel, Patrick J; Preston, Christopher; Duke, Stephen O

    2014-09-01

    Two major classes of herbicides include inhibitors of protoporphyrinogen oxidase (PPO) and phytoene desaturase (PDS). Plants can evolve resistance to PPO and PDS inhibitors via several mechanisms that include physical changes, resulting in reduced uptake, physiological changes, resulting in compartmentalization or altered translocation, and biochemical changes, resulting in enhanced metabolic degradation or alterations of protein structures, leading to loss of sensitivity to the herbicides. This review discusses the involvement of some of these mechanisms in the various cases of resistance to PDS- and PPO-inhibiting herbicides, and highlights unique aspects of target-site resistance to these herbicides.

  12. Melatonin directly interacts with cholesterol and alleviates cholesterol effects in dipalmitoylphosphatidylcholine monolayers.

    PubMed

    Choi, Youngjik; Attwood, Simon J; Hoopes, Matthew I; Drolle, Elizabeth; Karttunen, Mikko; Leonenko, Zoya

    2014-01-01

    Melatonin is a pineal hormone that has been shown to have protective effects in several diseases that are associated with cholesterol dysregulation, including cardiovascular disease, Alzheimer's disease, and certain types of cancers. Cholesterol is a major membrane constituent with both a structural and functional influence. It is also known that melatonin readily partitions into cellular membranes. We investigated the effects of melatonin and cholesterol on the structure and physical properties of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer as a simple membrane model using the Langmuir-Blodgett (L-B) monolayer technique and molecular dynamics (MD) simulations. We report that melatonin increases the area per lipid and elastic compressibility of the DPPC monolayer in a concentration dependent manner, while cholesterol has the opposite effect. When both melatonin and cholesterol were present in the monolayer, the compression isotherms showed normalization of the area per molecule towards that of the pure DPPC monolayer, thus indicating that melatonin counteracts and alleviates cholesterol's effects. Atomistic MD simulations of melatonin enriched DPPC systems correlate with our experimental findings and illustrate the structural effects of both cholesterol and melatonin. Our results suggest that melatonin is able to lessen the influence of cholesterol through two different mechanisms. Firstly, we have shown that melatonin has a fluidizing effect on monolayers comprising only lipid molecules. Secondly, we also observe that melatonin interacts directly with cholesterol. Our findings suggest a direct nonspecific interaction of melatonin may be a mechanism involved in reducing cholesterol associated membrane effects, thus suggesting the existence of a new mechanism of melatonin's action. This may have important biological relevance in addition to the well-known anti-oxidative and receptor binding effects. PMID:24651707

  13. Enzymatic Quantification of Cholesterol and Cholesterol Esters from Silicone Hydrogel Contact Lenses

    PubMed Central

    Pucker, Andrew D.; Thangavelu, Mirunalni

    2010-01-01

    Purpose. The purpose of this work was to develop an enzymatic method of quantification of cholesterol and cholesterol esters derived from contact lenses, both in vitro and ex vivo. Methods. Lotrafilcon B (O2 Optix; CIBA Vision, Inc., Duluth, GA) and galyfilcon A (Acuvue Advance; Vistakon, Inc., Jacksonville, FL) silicone hydrogel contact lenses were independently incubated in cholesterol oleate solutions varying in concentrations. After incubation, the lenses were removed and underwent two separate 2:1 chloroform-methanol extractions. After in vitro studies, 10 human subjects wore both lotrafilcon B and galyfilcon A contact lenses for 7 days. The lenses also underwent two separate 2:1 chloroform-methanol extractions. All in vitro and ex vivo samples were quantified with a cholesterol esterase enzymatic reaction. Results. Calibration curves from quantifications of in vitro contact lens samples soaked in successively decreasing concentrations of cholesterol oleate yielded coefficients of determination (R2) of 0.99 (lotrafilcon B) and 0.97 (galyfilcon A). For in vitro contact lens samples, galyfilcon A was associated with an average cholesterol oleate extraction of 39.85 ± 48.65 μg/lens, whereas lotrafilcon B was associated with 5.86 ± 3.36 μg/lens (P = 0.05) across both extractions and all incubation concentrations. For ex vivo contact lens samples, there was significantly more cholesterol and cholesterol esters deposited on galyfilcon A (5.77 ± 1.87 μg/lens) than on lotrafilcon B (2.03 ± 1.62 μg/lens; P = 0.0005). Conclusions. This is an efficient and simple method of quantifying total cholesterol extracted from silicone hydrogel contact lenses and, potentially, the meibum and/or tear film. Certain silicone hydrogel materials demonstrate more affinity for cholesterol and its esters than do others. PMID:20089871

  14. Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

    PubMed

    Zajoncová, L; Frébort, I; Luhová, L; Sebela, M; Galuszka, P; Pec, P

    1999-01-01

    Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed. PMID:10092944

  15. Human scavenger receptor class B type II (SR-BII) and cellular cholesterol efflux.

    PubMed Central

    Mulcahy, Jane V; Riddell, Dave R; Owen, James S

    2004-01-01

    Although studies in recombinant cells indicate that scavenger receptor class B, type I (SR-BI) can promote cholesterol efflux, investigations in transgenic mice overexpressing or deficient in SR-BI endorse its physiological function as selectively sequestering cholesteryl esters from high-density lipoproteins (HDLs). Less clear is the role of SR-BII, a splice variant of the SR-B gene that differs only in the C-terminal cytoplasmic domain. Here, we identify several putative signalling motifs in the C-terminus of human SR-BII, which are absent from SR-BI, and hypothesize that these motifs interact with signalling molecules to mobilize stored cholesteryl esters and/or promote the efflux of intracellular free cholesterol. 'Pull-down' assays using a panel of tagged SH3 (Src homology 3) domains showed that cytoplasmic SR-BII, but not cytoplasmic SR-BI, bound the SH3 domain of phospholipase C-gamma1; this interaction was not, however, detected under more physiological conditions. Specific anti-peptide antisera identified SR-BII in human monocyte/macrophage THP-1 cells and, in recombinant cells, revealed receptor localization to caveolae, a plasma membrane microdomain that concentrates signal-transducer molecules and acts as a conduit for cholesterol flux between cells and lipoproteins. Consistent with its caveolar localization, expression of human SR-BII in recombinant Chinese hamster ovary cells (CHO-SR-BII) was associated with increased HDL-mediated cholesterol efflux. Nevertheless, when CHO-SR-BII cells were pre-loaded with cholesteryl [(3)H]oleate and incubated with HDL, cholesteryl ester stores were not reduced compared with control cells. We conclude that although human SR-BII is expressed by macrophages, contains cytoplasmic signalling motifs and localizes to caveolae, its ability to stimulate cholesterol efflux does not reflect enhanced hydrolysis of stored cholesteryl esters. PMID:14570588

  16. Genetic Analysis of Digestive Physiology Using Fluorescent Phospholipid Reporters

    NASA Astrophysics Data System (ADS)

    Farber, Steven A.; Pack, Michael; Ho, Shiu-Ying; Johnson, Iain D.; Wagner, Daniel S.; Dosch, Roland; Mullins, Mary C.; Hendrickson, H. Stewart; Hendrickson, Elizabeth K.; Halpern, Marnie E.

    2001-05-01

    Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

  17. Cell Wall Amine Oxidases: New Players in Root Xylem Differentiation under Stress Conditions.

    PubMed

    Ghuge, Sandip A; Tisi, Alessandra; Carucci, Andrea; Rodrigues-Pousada, Renato A; Franchi, Stefano; Tavladoraki, Paraskevi; Angelini, Riccardo; Cona, Alessandra

    2015-01-01

    Polyamines (PAs) are aliphatic polycations present in all living organisms. A growing body of evidence reveals their involvement as regulators in a variety of physiological and pathological events. They are oxidatively deaminated by amine oxidases (AOs), including copper amine oxidases (CuAOs) and flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). The biologically-active hydrogen peroxide (H₂O₂) is a shared compound in all of the AO-catalyzed reactions, and it has been reported to play important roles in PA-mediated developmental and stress-induced processes. In particular, the AO-driven H₂O₂ biosynthesis in the cell wall is well known to be involved in plant wound healing and pathogen attack responses by both triggering peroxidase-mediated wall-stiffening events and signaling modulation of defense gene expression. Extensive investigation by a variety of methodological approaches revealed high levels of expression of cell wall-localized AOs in root xylem tissues and vascular parenchyma of different plant species. Here, the recent progresses in understanding the role of cell wall-localized AOs as mediators of root xylem differentiation during development and/or under stress conditions are reviewed. A number of experimental pieces of evidence supports the involvement of apoplastic H₂O₂ derived from PA oxidation in xylem tissue maturation under stress-simulated conditions. PMID:27135338

  18. The copper-iron connection in biology: Structure of the metallo-oxidase Fet3p

    SciTech Connect

    Taylor, A. B.; Stoj, C. S.; Ziegler, L.; Kosman, D. J.; Hart, P. J.

    2005-10-17

    Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase.

  19. Pathological changes in platelet histamine oxidases in atopic eczema

    PubMed Central

    Ionescu, Gruia

    1993-01-01

    Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu2+) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe2+) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase. PMID:18475554

  20. CHOBIMALT: A Cholesterol-Based Detergent†

    PubMed Central

    Howell, Stanley C.; Mittal, Ritesh; Huang, Lijun; Travis, Benjamin; Breyer, Richard M.; Sanders, Charles R.

    2010-01-01

    Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins, but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3–4μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210 ± 30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1. PMID:20919740

  1. CHOBIMALT: a cholesterol-based detergent.

    PubMed

    Howell, Stanley C; Mittal, Ritesh; Huang, Lijun; Travis, Benjamin; Breyer, Richard M; Sanders, Charles R

    2010-11-01

    Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3−4 μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1.

  2. Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

    PubMed

    Barrientos, Genaro; Llanos, Paola; Hidalgo, Jorge; Bolaños, Pura; Caputo, Carlo; Riquelme, Alexander; Sánchez, Gina; Quest, Andrew F G; Hidalgo, Cecilia

    2015-01-01

    Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction (E-C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca(2+) transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na(+)/K(+)-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47(phox) and gp91(phox)), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E-C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

  3. Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

    2014-03-01

    Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

  4. Nox family NADPH oxidases: Molecular mechanisms of activation.

    PubMed

    Brandes, Ralf P; Weissmann, Norbert; Schröder, Katrin

    2014-11-01

    NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS). Numerous homologue-specific mechanisms control the activity of this enzyme family involving calcium, free fatty acids, protein-protein interactions, intracellular trafficking, and posttranslational modifications such as phosphorylation, acetylation, or sumoylation. After a brief review on the classic pathways of Nox activation, this article will focus on novel mechanisms of homologue-specific activity control and on cell-specific aspects which govern Nox activity. From these findings of the recent years it must be concluded that the activity control of Nox enzymes is much more complex than anticipated. Moreover, depending on the cellular activity state, Nox enzymes are selectively activated or inactivated. The complex upstream signaling aspects of these events make the development of "intelligent" Nox inhibitors plausible, which selectively attenuate disease-related Nox-mediated ROS formation without altering physiological signaling ROS. This approach might be of relevance for Nox-mediated tissue injury in ischemia-reperfusion and inflammation and also for chronic Nox overactivation as present in cancer initiation and cardiovascular disease.

  5. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues.

    PubMed

    Jiménez-Quesada, María J; Traverso, José Á; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen-pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed.

  6. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues

    PubMed Central

    Jiménez-Quesada, María J.; Traverso, José Á.; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen–pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed. PMID:27066025

  7. The glycemic index: physiological significance.

    PubMed

    Esfahani, Amin; Wong, Julia M W; Mirrahimi, Arash; Srichaikul, Korbua; Jenkins, David J A; Kendall, Cyril W C

    2009-08-01

    The glycemic index (GI) is a physiological assessment of a food's carbohydrate content through its effect on postprandial blood glucose concentrations. Evidence from trials and observational studies suggests that this physiological classification may have relevance to those chronic Western diseases associated with overconsumption and inactivity leading to central obesity and insulin resistance. The glycemic index classification of foods has been used as a tool to assess potential prevention and treatment strategies for diseases where glycemic control is of importance, such as diabetes. Low GI diets have also been reported to improve the serum lipid profile, reduce C-reactive protein (CRP) concentrations, and aid in weight control. In cross-sectional studies, low GI or glycemic load diets (mean GI multiplied by total carbohydrate) have been associated with higher levels of high-density lipoprotein cholesterol (HDL-C), with reduced CRP concentrations, and, in cohort studies, with decreased risk of developing diabetes and cardiovascular disease. In addition, some case-control and cohort studies have found positive associations between dietary GI and risk of various cancers, including those of the colon, breast, and prostate. Although inconsistencies in the current findings still need to be resolved, sufficient positive evidence, especially with respect to renewed interest in postprandial events, suggests that the glycemic index may have a role to play in the treatment and prevention of chronic diseases.

  8. In vitro antimalarial and xanthine oxidase inhibition of 2-Aminoanthraquinone.

    PubMed

    Rauf, Abdur; Khan, Rehan; Khan, Haroon; Jehan, Noor; Akram, Mohammad; Ahmad, Zarka; Muhammad, Naveed; Farooq, Umar; Khan, Ajmal

    2016-03-01

    In the present research study 2-Aminoanthraquinone were scrutinized for their antimalarial and Xanthine oxidase inhibitor potential. It demonstrated marked concentration dependent antimalarial activity with maximum effect of 89.06% and with IC50 of 34.17 µM. Regarding Xanthine oxidase inhibitor activity, it evoked significant effect with 57.45% activity with IC50 value of 81.57.19 μM. In conclusion, 2-Aminoanthraquinone showed potent antimalarial and xanthine oxidase inhibitory activity. PMID:27087090

  9. Cholesterol granuloma of the breast mimicking malignancy

    PubMed Central

    Khan, Roobina; Narula, Varsha; Jain, Anshu; Maheshwari, Veena

    2013-01-01

    Cholesterol granuloma of the breast is an unusual non-neoplastic condition which frequently resembles malignancy clinically as well as radiologically. We herein report a case of a 28-year-old woman who presented with a lump in the upper outer quadrant of right breast. Physical examination and ultrasonography strongly suggested a carcinomatous lesion, but histopathological examination confirmed the diagnosis to be cholestrol granulomas. This article highlights the perplexing clinical presentation of cholesterol granulomas and affirms the role of histopathological examination in diagnosing these lesions correctly. PMID:23925685

  10. Thyroidal regulation of substrate kinetics properties of cytochrome oxidase in rat liver mitochondria.

    PubMed

    Modi, Hiren R; Katyare, Surendra S; Patel, Samir P

    2008-07-01

    Effects of thyroidectomy (T(x)) and subsequent treatment with 3,5,3'-triiodothyronine (T(3)), and combined treatment (T(R)) with T(3) + thyroxine (T(4)) on substrate kinetics properties of cytochrome oxidase of rat liver mitochondria were examined. T(x) resulted in lowering of cytochromes content with decrease in the enzyme activity, and K(m) and V(max). T(3) and T(R) regimens restored the cytochromes contents and the V(max) values to normal. In control, T(3) and T(R) groups the enzyme activity resolved in two kinetic components; in T(x) group three kinetic components were evident. The K(m) values for all components decreased significantly in the experimental groups with concomitant increase in catalytic efficiency, K(cat)/K(m). Significant alterations in the contents of total phospholipid and of cholesterol were noted while the changes in the phospholipids composition were only of restricted nature. Regression analysis revealed that total phospholipid, cholesterol and phosphatidylcholine, phosphatidylethanolamine play significant role in fine tuning the enzyme activity. PMID:23105769

  11. Crystal Structure of a Two-domain Multicopper Oxidase

    PubMed Central

    Lawton, Thomas J.; Sayavedra-Soto, Luis A.; Arp, Daniel J.; Rosenzweig, Amy C.

    2009-01-01

    The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 Å resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases. PMID:19224923

  12. A new framework for reverse cholesterol transport: Non-biliary contributions to reverse cholesterol transport

    PubMed Central

    Temel, Ryan E; Brown, J Mark

    2010-01-01

    Reduction of low-density lipoprotein-cholesterol through statin therapy has only modestly decreased coronary heart disease (CHD)-associated mortality in developed countries, which has prompted the search for alternative therapeutic strategies for CHD. Major efforts are now focused on therapies that augment high-density lipoprotein (HDL)-mediated reverse cholesterol transport (RCT), and ultimately increase the fecal disposal of cholesterol. The process of RCT has long been thought to simply involve HDL-mediated delivery of peripheral cholesterol to the liver for biliary excretion out of the body. However, recent studies have revealed a novel pathway for RCT that does not rely on biliary secretion. This non-biliary pathway rather involves the direct excretion of cholesterol by the proximal small intestine. Compared to RCT therapies that augment biliary sterol loss, modulation of non-biliary fecal sterol loss through the intestine is a much more attractive therapeutic strategy, given that excessive biliary cholesterol secretion can promote gallstone formation. However, we are at an early stage in understanding the molecular mechanisms regulating the non-biliary pathway for RCT, and much additional work is required in order to effectively target this pathway for CHD prevention. The purpose of this review is to discuss our current understanding of biliary and non-biliary contributions to RCT with particular emphasis on the possibility of targeting the intestine as an inducible cholesterol secretory organ. PMID:21157970

  13. Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export

    PubMed Central

    Li, Jian; Pfeffer, Suzanne R

    2016-01-01

    LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly. DOI: http://dx.doi.org/10.7554/eLife.21635.001 PMID:27664420

  14. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain

    PubMed Central

    Lund, Erik G.; Guileyardo, Joseph M.; Russell, David W.

    1999-01-01

    The turnover of cholesterol in the brain is thought to occur via conversion of excess cholesterol into 24S-hydroxycholesterol, an oxysterol that is readily secreted from the central nervous system into the plasma. To gain molecular insight into this pathway of cholesterol metabolism, we used expression cloning to isolate cDNAs that encode murine and human cholesterol 24-hydroxylases. DNA sequence analysis indicates that both proteins are localized to the endoplasmic reticulum, share 95% identity, and represent a new cytochrome P450 subfamily (CYP46). When transfected into cultured cells, the cDNAs produce an enzymatic activity that converts cholesterol into 24S-hydroxycholesterol, and to a lesser extent, 25-hydroxycholesterol. The cholesterol 24-hydroxylase gene contains 15 exons and is located on human chromosome 14q32.1. Cholesterol 24-hydroxylase is expressed predominantly in the brain as judged by RNA and protein blotting. In situ mRNA hybridization and immunohistochemistry localize the expression of this P450 to neurons in multiple subregions of the brain. The concentrations of 24S-hydroxycholesterol in serum are low in newborn mice, reach a peak between postnatal days 12 and 15, and thereafter decline to baseline levels. In contrast, cholesterol 24-hydroxylase protein is first detected in the brain of mice at birth and continues to accumulate with age. We conclude that the cloned cDNAs encode cholesterol 24-hydroxylases that synthesize oxysterols in neurons of the brain and that secretion of 24S-hydroxycholesterol from this tissue in the mouse is developmentally regulated. PMID:10377398

  15. Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice.

    PubMed

    Bura, Kanwardeep S; Lord, Caleb; Marshall, Stephanie; McDaniel, Allison; Thomas, Gwyn; Warrier, Manya; Zhang, Jun; Davis, Matthew A; Sawyer, Janet K; Shah, Ramesh; Wilson, Martha D; Dikkers, Arne; Tietge, Uwe J F; Collet, Xavier; Rudel, Lawrence L; Temel, Ryan E; Brown, J Mark

    2013-06-01

    Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BI(hApoCIII-ApoAIV-Tg)). SR-BI(hApoCIII-ApoAIV-Tg) mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BI(hApoCIII-ApoAIV-Tg) mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BI(hApoCIII-ApoAIV-Tg) mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

  16. Characterization of polyphenol oxidase and peroxidase and influence on browning of cold stored strawberry fruit.

    PubMed

    Chisari, Marco; Barbagallo, Riccardo N; Spagna, Giovanni

    2007-05-01

    Polyphenol oxidase and peroxidase were extracted from two different varieties of strawberry fruit (Fragaria x ananassa D, cv. 'Elsanta' and Fragaria vesca L, cv. 'Madame Moutot') and characterized using reliable spectrophotometric methods. In all cases, the enzymes followed Michaelis-Menten kinetics, showing different values of peroxidase kinetics parameters between the two cultivars: Km = 50.68 +/- 2.42 mM ('Elsanta') and 18.18 +/- 8.79 mM ('Madame Moutot') mM and Vmax = 0.14 +/- 0.03 U/g ('Elsanta') and 0.05 +/- 0.01 U/g ('Madame Moutot'). The physiological pH of fruit at the red ripe stage negatively affected the expression of both oxidases, except polyphenol oxidase from 'Madame Moutot' that showed the highest residual activity (68% of the maximum). Peroxidase from both cultivars was much more thermolable as compared with PPO, losing over 60% of relative activity already after 60 min of incubation at 40 degrees C. The POD activation energy was much lower than the PPO activation energy (DeltaE = 97.5 and 57.8 kJ mol-1 for 'Elsanta' and 'Madame Moutot', respectively). Results obtained from d-glucose and d-fructose inhibition tests evidenced a decreasing course of PPO and POD activities from both cultivars as the sugar concentration in the assay medium increased. Changes in CIE L*, a*, b*, chroma, and hue angle values were taken as a browning index of the samples during storage at 4 degrees C. A decrease in L* was evident in both cultivars but more marked in 'Elsanta'. PPO and POD activities from cv. 'Elsanta' were very well-correlated with the parameter L* (r2=0.86 and 0.89, respectively) and hue angle (r2=0.85 and 0.93, respectively). According to these results, the browning of the fruit seemed to be in relation to both oxidase activities.

  17. Characterization of polyphenol oxidase and peroxidase and influence on browning of cold stored strawberry fruit.

    PubMed

    Chisari, Marco; Barbagallo, Riccardo N; Spagna, Giovanni

    2007-05-01

    Polyphenol oxidase and peroxidase were extracted from two different varieties of strawberry fruit (Fragaria x ananassa D, cv. 'Elsanta' and Fragaria vesca L, cv. 'Madame Moutot') and characterized using reliable spectrophotometric methods. In all cases, the enzymes followed Michaelis-Menten kinetics, showing different values of peroxidase kinetics parameters between the two cultivars: Km = 50.68 +/- 2.42 mM ('Elsanta') and 18.18 +/- 8.79 mM ('Madame Moutot') mM and Vmax = 0.14 +/- 0.03 U/g ('Elsanta') and 0.05 +/- 0.01 U/g ('Madame Moutot'). The physiological pH of fruit at the red ripe stage negatively affected the expression of both oxidases, except polyphenol oxidase from 'Madame Moutot' that showed the highest residual activity (68% of the maximum). Peroxidase from both cultivars was much more thermolable as compared with PPO, losing over 60% of relative activity already after 60 min of incubation at 40 degrees C. The POD activation energy was much lower than the PPO activation energy (DeltaE = 97.5 and 57.8 kJ mol-1 for 'Elsanta' and 'Madame Moutot', respectively). Results obtained from d-glucose and d-fructose inhibition tests evidenced a decreasing course of PPO and POD activities from both cultivars as the sugar concentration in the assay medium increased. Changes in CIE L*, a*, b*, chroma, and hue angle values were taken as a browning index of the samples during storage at 4 degrees C. A decrease in L* was evident in both cultivars but more marked in 'Elsanta'. PPO and POD activities from cv. 'Elsanta' were very well-correlated with the parameter L* (r2=0.86 and 0.89, respectively) and hue angle (r2=0.85 and 0.93, respectively). According to these results, the browning of the fruit seemed to be in relation to both oxidase activities. PMID:17407312

  18. Growth of calcium hydroxyapatite (Ca-HAp) on cholesterol and cholestanol crystals from a simulated body fluid: A possible insight into the pathological calcifications associated with atherosclerosis.

    PubMed

    Laird, Dougal F; Mucalo, Michael R; Yokogawa, Yoshiyuki

    2006-03-15

    An experimental study into calcium phosphate (CP) nucleation and growth on cholesterol and cholestanol surfaces from a supersaturated simulated body fluid (SBF) is presented with the overall aim of gaining some fundamental insights into the pathological calcifications associated with atherosclerosis. Soaking of pressed cholesterol disks at physiological temperature in SBF solutions was found to lead to CP nucleation and growth if the disks were surface roughened and if an SBF with concentrations of the calcium and hydrogen phosphate ions at 2.25x physiological concentrations was used. The CP phase deposited was shown via SEM micrographs to possess a florette type morphology akin to that observed in earlier reported studies. The use of recrystallised cholesterol and cholestanol microcrystals as substrates for soaking in SBF facilitated the observation of CP deposition. In general, cholesterol recrystallised from polar solvents like 95% ethanol as a cholesterol monohydrate phase which was a better substrate for CP growth than cholesterol recrystallised from more non-polar solvents (e.g., benzene) which produced anhydrous cholesterol phases. CP was also observed to form on recrystallised cholestanol microcrystals, a molecule closely related to cholesterol. Inductively coupled plasma optical emission spectrometry (ICP-OES) data gave confirmation that Ca:P mole ratios of the grown CP were 1.3-1.5 suggesting a mixed phase of octacalcium phosphate (OCP) and Ca-deficient HAp and that the CP coating grows (with time of soaking) on the substrates after nucleation in the SBF growth medium. Infrared (IR) spectra of the extracted coatings from the cholesterol substrates confirmed that the CP phase deposited is a semi crystalline HAp with either carbonate substituted into its structure or else co-deposited as calcium carbonate. Soaking experiments involving modified cholesterol substrates in which the OH group in the molecule was replaced with the oleiyl or phosphonate group

  19. [THE SPIRIT CHOLESTEROL, BIOLOGICA L ROLE AT STAGES OF PHYLOGENESIS, MECHANISMS OF INHIBITION OF SYNTHESIS OF STEROL BY STATINS, FACTORS OF PHARMACOGENOMICS AND DIAGNOSTIC SIGNIFICANCE OF CHOLESTEROL OF LIPOPROTEINS OF LOW DENSITY].

    PubMed

    Titov, V N; Kotlovskii, M Yu; Pokrovskii, A A; Kotlovskaia, O S; Osedko, A V; Titova, N M; Kotlovskii, Yu V; Digaii, A M

    2015-04-01

    The hypolipidemic effect of statins is realized by inhibition of synthesis of local pool of cholesterol spirit in endoplasmic net of hepatocytes. The cholesterol spirit covers all hydrophobic medium of triglycerides with polar mono layer of phosphatidylcholines and cholesterol spirit prior to secretion of lipoproteins of very low density into hydrophilic medium. The lesser mono layer between lipase enzyme and triglycerides substrate contains of cholesterol spirit the higher are the parameters of hydrolysis of palmitic and oleic lipoproteins of very low density. The sequence of effect of statins is as follows: blocking of synthesis in hepatocytes and decreasing of content of unesterified cholesterol spirit in blood plasma; activation of hydrolysis of triglycerides in palmitic and oleic lipoproteins of very low density; formation of ligand lipoproteins of very low density and their absorption by cells by force of apoB-100 endocytosis; decreasing in blood of content of polyenoic fatty acids, equimolar esterified by cholesterol spirit, polyethers of cholesterol spirit and decreasing of level of cholesterol spirit-lipoproteins of very low density. There is no way to eliminate aphysiological effect of disordered biological function of trophology (nutrition) on metabolism of fatty acids in population by means of pharmaceuticals intake. It is necessary to eliminate aphysiological effect of environment. To decrease rate of diseases of cardiovascular system one has to decrease in food content of saturated fatty acids and in the first instance palmitic saturated fatty acid, trans-form fatty acid, palmitoleic fatty acids up to physiological values and increase to the same degree the content of polyenoic fatty acids. The saturated fatty acids block absorption of polyenoic fatty acids by cells. The atherosclerosis is a deficiency of polyenoic fatty acids under surplus of palmitic saturated fatty acid. PMID:26189285

  20. Is Xanthine oxidase activity in polycystic ovary syndrome associated with inflammatory and cardiovascular risk factors?

    PubMed

    Isık, Hatice; Aynıoglu, Oner; Tımur, Hakan; Sahbaz, Ahmet; Harma, Muge; Can, Murat; Guven, Berrak; Alptekin, Husnu; Kokturk, Furuzan

    2016-08-01

    The aim of this study is to examine women with polycystic ovary syndrome (PCOS) to determine the relationship between xanthine oxidase (XO) and oxidative stress, inflammatory status, and various clinical and biochemical parameters. In this cross-sectional study a total of 83 women including 45 PCOS patients and 38 healthy women were enrolled. We collected blood samples for XO and superoxide dismutase (SOD) activity, hormone levels, cholesterol values, and inflammatory markers. Body mass index (BMI) , waist-to-hip ratio (WHR), and blood pressure were assessed. Blood samples were taken for hormonal levels, cholesterol levels, fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostatic model assessment-insulin resistance (HOMA-IR) index, quantitative insulin sensitivity check index (QUICKI), C-reactive protein (CRP), white blood cell and neutrophil counts, XO and SOD activities. The basal hormone levels, triglyceride (TG) levels, TG/HDL-C (high density lipoprotein-cholesterol) ratios FPG, FPI and HOMA-IR levels were higher in PCOS patients compared to controls (p<0.05). Platelet and plateletcrit (PCT) values, CRP, and XO activity were significantly increased, however SOD activity was decreased in PCOS patients (p<0.001). XO activity was positively correlated with LH/FSH and TG/HDL ratios, CRP, PCT, FPG, FPI, and HOMA-IR, and negatively correlated with QUICKI levels. In conclusion, XO is a useful marker to assess oxidative stress in PCOS patients. Positive correlations between XO and inflammatory markers and cardiovascular disease risk factors suggest that XO plays an important role in the pathogenesis of PCOS and its metabolic complications. PMID:27295433

  1. The cross-talk of LDL-cholesterol with cell motility: insights from the Niemann Pick Type C1 mutation and altered integrin trafficking.

    PubMed

    Hoque, Monira; Rentero, Carles; Conway, James R; Murray, Rachael Z; Timpson, Paul; Enrich, Carlos; Grewal, Thomas

    2015-01-01

    Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility.

  2. The ATP-binding Cassette Transporter-2 (ABCA2) Regulates Cholesterol Homeostasis and Low-density Lipoprotein Receptor Metabolism in N2a Neuroblastoma Cells

    PubMed Central

    Davis, Warren

    2011-01-01

    The ATP-binding cassette transporter-2 (ABCA2) has been identified as a possible regulator of lipid metabolism. ABCA2 is most highly expressed in the brain but its effects on cholesterol homeostasis in neuronal-type cells have not been characterized. It is important to study the role of ABCA2 in regulating cholesterol homeostasis in neuronal-type cells because ABCA2 has been identified as a possible genetic risk factor for Alzheimer’s disease. In this study, the effects of ABCA2 expression on cholesterol homeostasis were examined in mouse N2a neuroblastoma cells. ABCA2 reduced total, free- and esterified cholesterol levels as well as membrane cholesterol but did not perturb cholesterol distribution in organelle or lipid raft compartments. ABCA2 did not modulate de novo cholesterol biosynthesis from acetate. Cholesterol trafficking to the plasma membrane was not affected by ABCA2 but efflux to the physiological acceptor ApoE3 and mobilization of plasma membrane cholesterol to the endoplasmic reticulum for esterification were reduced by ABCA2. ABCA2 reduced esterification of serum and low-density lipoprotein-derived cholesterol but not 25-hydroxycholesterol. ABCA2 decreased low-density lipoprotein receptor (LDLR) mRNA and protein levels and increased its turnover rate. The surface expression of LDLR as well as the uptake of fluroresecent DiI-LDL was also reduced by ABCA2. Reduction of endogenous ABCA2 expression by RNAi treatment of N2a cells and rat primary cortical neurons produced the opposite effects of over-expression of ABCA2, increasing LDLR protein levels. This report identifies ABCA2 as a key regulator of cholesterol homeostasis and LDLR metabolism in neuronal cells. PMID:21810484

  3. Regulation of biliary cholesterol secretion. Functional relationship between the canalicular and sinusoidal cholesterol secretory pathways in the rat.

    PubMed Central

    Nervi, F; Marinović, I; Rigotti, A; Ulloa, N

    1988-01-01

    The functional interrelationship between biliary cholesterol secretion, sinusoidal lipoprotein cholesterol secretion and bile salt synthesis was studied in the rat. Diosgenin, fructose, and colestipol in the diet were used to, respectively, influence biliary cholesterol output, VLDL production and bile salt synthesis. In the acute bile fistula rat, biliary cholesterol output was 700% increased by diosgenin and 50% decreased by fructose. In the rats fed both diosgenin and fructose, biliary cholesterol secretion was increased only by approximately 200%, whereas biliary bile salts and phospholipid outputs were unchanged. In the isolated perfused liver, VLDL-cholesterol output was 50% reduced by diosgenin alone, but was unchanged following feeding of diosgenin plus fructose. However, the livers of rats fed diosgenin plus fructose exhibited a 700% increase in VLDL-triglyceride production and a 200% increase in VLDL-cholesterol output. A significant reciprocal relationship between VLDL-cholesterol secretion and the coupling ratio of cholesterol to bile salts in bile was observed. Colestipol added to the diet maintained both sinusoidal and biliary cholesterol outputs within the normal range. In the chronic bile fistula rat, colestipol increased bile salt synthesis by 100% while diosgenin and fructose diets had no effect. Similarly, the addition of fructose to the colestipol diet did not decrease bile salt synthesis. These data suggest a reciprocal relationship between biliary cholesterol secretion and hepatic secretion of cholesterol as VLDL particles. The free cholesterol pool used for bile salt synthesis seems functionally unrelated to the pool from which VLDL-cholesterol and biliary cholesterol originate. These findings support the idea that metabolic compartmentalization of hepatic cholesterol is a major determinant of the quantity of cholesterol available for recruitment by the bile salt-dependent biliary cholesterol secretory mechanism. PMID:3198756

  4. Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment.

    PubMed

    Báez-Pagán, Carlos A; Del Hoyo-Rivera, Natalie; Quesada, Orestes; Otero-Cruz, José David; Lasalde-Dominicci, José A

    2016-08-01

    The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane. PMID:27116687

  5. Scavenger receptor BI: a multi-purpose player in cholesterol and steroid metabolism.

    PubMed

    Hoekstra, Menno; Van Berkel, Theo-Jc; Van Eck, Miranda

    2010-12-21

    Scavenger receptor class B type I (SR-BI) is an important member of the scavenger receptor family of integral membrane glycoproteins. This review highlights studies in SR-BI knockout mice, which concern the role of SR-BI in cholesterol and steroid metabolism. SR-BI in hepatocytes is the sole molecule involved in selective uptake of cholesteryl esters from high-density lipoprotein (HDL). SR-BI plays a physiological role in binding and uptake of native apolipoprotein B (apoB)-containing lipoproteins by hepatocytes, which identifies SR-BI as a multi-purpose player in lipid uptake from the blood circulation into hepatocytes in mice. In adrenocortical cells, SR-BI mediates the selective uptake of HDL-cholesteryl esters, which is efficiently coupled to the synthesis of glucocorticoids (i.e. corticosterone). SR-BI knockout mice suffer from adrenal glucocorticoid insufficiency, which suggests that functional SR-BI protein is necessary for optimal adrenal steroidogenesis in mice. SR-BI in macrophages plays a dual role in cholesterol metabolism as it is able to take up cholesterol associated with HDL and apoB-containing lipoproteins and can possibly facilitate cholesterol efflux to HDL. Absence of SR-BI is associated with thrombocytopenia and altered thrombosis susceptibility, which suggests a novel role for SR-BI in regulating platelet number and function in mice. Transgenic expression of cholesteryl ester transfer protein in humanized SR-BI knockout mice normalizes hepatic delivery of HDL-cholesteryl esters. However, other pathologies associated with SR-BI deficiency, i.e. increased atherosclerosis susceptibility, adrenal glucocorticoid insufficiency, and impaired platelet function are not normalized, which suggests an important role for SR-BI in cholesterol and steroid metabolism in man. In conclusion, generation of SR-BI knockout mice has significantly contributed to our knowledge of the physiological role of SR-BI. Studies using these mice have identified SR-BI as a

  6. The Hippo pathway, p53 and cholesterol.

    PubMed

    Aylon, Yael; Oren, Moshe

    2016-09-01

    ASBTRACT Increased rates of cholesterol and lipid synthesis have long been recognized as important aspects of the metabolic rewiring that occurs during cancerous transformation. Many genes encoding enzymes involved in cholesterol and fatty acid biogenesis are transcriptional targets of the sterol regulatory element-binding proteins (SREBPs). The SREBPs act as a hub for metabolic and proliferation-related signals; their activity is the focus of a tug-of-war between tumor suppressors, who generally inhibit SREBP function, and oncogenes, who often promote, and rely on, SREBP activity. The Hippo pathway plays a central role in coordinating cell proliferation and organ size, whereas p53 is a crucial tumor suppressor that maintains metabolic homeostasis and orchestrates cellular stress responses. Together, the Hippo and p53 signaling pathways cooperate on multiple levels to fine-tune SREPB activity and regulate cholesterol/lipid levels. Cholesterol biosynthesis inhibitors such as statins are appealing conceptually, but have yet to show an indisputable effect on cancer development. Fortunately, the complex regulation surrounding the Hippo-p53-SREBP network potentially provides a broad interface for additional novel cancer-targeting interventions. PMID:27419353

  7. Genetic defects in postsqualene cholesterol biosynthesis.

    PubMed

    Moebius, F F; Fitzky, B U; Glossmann, H

    2000-04-01

    In humans and mice, four different genetic defects in the nine biosynthetic steps from lanosterol to cholesterol have been identified. They impair the activity of a putative C3-sterol dehydrogenase (Nshdl, X-linked dominant bare patches/striated mutation in mice), the sterol delta 8-delta 7 isomerase/EBP (Ebp, X-linked dominant tattered mutation in mice; chondrodysplasia punctata (CDPX2) in humans), the delta 24-sterol reductase (autosomal recessive desmosterolosis) and the delta 7-sterol reductase (DHCR7 gene, autosomal recessive Smith-Lemli-Opitz syndrome in humans). These inborn errors in postsqualene cholesterol metabolism result in dysmorphogenetic syndromes of variable severity. The X-linked dominant mutations result in mosaicism in females, as a result of X-inactivation, and midgestational lethality in males. The mechanisms by which the depletion of cholesterol or the accumulation of intermediates impair morphogenetic programs are unclear. So far, no cellular processes that require an intact cholesterol biosynthetic pathway have been identified, although the morphogenetic hedgehog-patched signaling cascade is a candidate. PMID:10707051

  8. Mitochondria, cholesterol and cancer cell metabolism.

    PubMed

    Ribas, Vicent; García-Ruiz, Carmen; Fernández-Checa, José C

    2016-12-01

    Given the role of mitochondria in oxygen consumption, metabolism and cell death regulation, alterations in mitochondrial function or dysregulation of cell death pathways contribute to the genesis and progression of cancer. Cancer cells exhibit an array of metabolic transformations induced by mutations leading to gain-of-function of oncogenes and loss-of-function of tumor suppressor genes that include increased glucose consumption, reduced mitochondrial respiration, increased reactive oxygen species generation and cell death resistance, all of which ensure cancer progression. Cholesterol metabolism is disturbed in cancer cells and supports uncontrolled cell growth. In particular, the accumulation of cholesterol in mitochondria emerges as a molecular component that orchestrates some of these metabolic alterations in cancer cells by impairing mitochondrial function. As a consequence, mitochondrial cholesterol loading in cancer cells may contribute, in part, to the Warburg effect stimulating aerobic glycolysis to meet the energetic demand of proliferating cells, while protecting cancer cells against mitochondrial apoptosis due to changes in mitochondrial membrane dynamics. Further understanding the complexity in the metabolic alterations of cancer cells, mediated largely through alterations in mitochondrial function, may pave the way to identify more efficient strategies for cancer treatment involving the use of small molecules targeting mitochondria, cholesterol homeostasis/trafficking and specific metabolic pathways. PMID:27455839

  9. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  10. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

    PubMed

    Peng, Zeyu; Dittmer, Neal T; Lang, Minglin; Brummett, Lisa M; Braun, Caroline L; Davis, Lawrence C; Kanost, Michael R; Gorman, Maureen J

    2015-04-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  11. Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

    PubMed Central

    Peng, Zeyu; Dittmer, Neal T.; Lang, Minglin; Brummett, Lisa M.; Braun, Caroline L.; Davis, Lawrence C.; Kanost, Michael R.; Gorman, Maureen J.

    2015-01-01

    Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surpring because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism. PMID:25701385

  12. Free-cholesterol-mediated autophagy of ORMDL1 stimulates sphingomyelin biosynthesis.

    PubMed

    Wang, Shuhui; Robinet, Peggy; Smith, Jonathan D; Gulshan, Kailash

    2015-01-01

    Cholesterol confers unique biophysical properties to the plasma membrane bilayer that are essential for maintaining optimal membrane fluidity, which in turn regulate multiple physiological functions required to promote cellular integrity and viability. Conversely, excessive cholesterol causes pathological conditions such as atherosclerosis that can lead to heart attacks. Human atheroma macrophages carry a large burden of free cholesterol (FC) in addition to cholesterol esters. It is recognized that sterols can modulate the levels of other lipids to attain lipid homeostasis; thus, excess FC may play a role in modulating compensatory sphingolipid pathways. Recent studies have shown that excess lipids can cause ER stress and apoptosis. In contrast, autophagy may play a protective role by clearing excess lipids from macrophage foam cell lipid droplets. Interestingly, a macrophage study using a TLR4-specifc agonist showed that de novo sphingolipid biosynthesis is essential for autophagy induction, suggesting links between sphingolipid biosynthesis and autophagy. While the role of autophagy in removing excess lipids has been the focus of many studies, its role in fine-tuning cellular lipid homeostasis remains largely unexplored. PMID:26042659

  13. Free-cholesterol-mediated autophagy of ORMDL1 stimulates sphingomyelin biosynthesis

    PubMed Central

    Wang, Shuhui; Robinet, Peggy; Smith, Jonathan D; Gulshan, Kailash

    2015-01-01

    Cholesterol confers unique biophysical properties to the plasma membrane bilayer that are essential for maintaining optimal membrane fluidity, which in turn regulate multiple physiological functions required to promote cellular integrity and viability. Conversely, excessive cholesterol causes pathological conditions such as atherosclerosis that can lead to heart attacks. Human atheroma macrophages carry a large burden of free cholesterol (FC) in addition to cholesterol esters. It is recognized that sterols can modulate the levels of other lipids to attain lipid homeostasis; thus, excess FC may play a role in modulating compensatory sphingolipid pathways. Recent studies have shown that excess lipids can cause ER stress and apoptosis. In contrast, autophagy may play a protective role by clearing excess lipids from macrophage foam cell lipid droplets. Interestingly, a macrophage study using a TLR4-specifc agonist showed that de novo sphingolipid biosynthesis is essential for autophagy induction, suggesting links between sphingolipid biosynthesis and autophagy. While the role of autophagy in removing excess lipids has been the focus of many studies, its role in fine-tuning cellular lipid homeostasis remains largely unexplored. PMID:26042659

  14. Hypolipidemic effects of HVC1 in a high cholesterol diet-induced rat model of hyperlipidemia

    PubMed Central

    Kim, Chae-Yun; Chung, Kyung-Sook; Cheon, Se-Yun; Lee, Kyungjin; Ham, Inhye; Choi, Ho-Young; Cho, Yong Baik; Cho, Byoung-Heon; Mok, So Youn; An, Hyo-Jin

    2016-01-01

    HVC1, a novel formation containing four herbs, was developed and its hypolipidemic effects in rats with high cholesterol diet (HCD)-induced hyperlipidemia were investigated. The rats were given a HCD for 8 weeks. The HVC1-treated groups were orally administered HVC1 at doses of 10, 50 or 250 mg/kg, respectively, and the simvastatin group was treated at a dose of 10 mg/kg. The normal diet and HCD control groups were administered with physiological saline. Oral administration of HVC1 (10, 50 or 250 mg/kg) significantly reduced the body weight of rats with hyperlipidemia and regulated the total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels in the serum. In addition, tissue analysis revealed that lipid accumulation in the liver and aorta was reduced by HVC1 administration. Furthermore, HVC1 significantly reduced the mRNA expression of peroxisome proliferator-activated receptor-γ, 3-hydroxy-3-methylglutaryl-CoA reductase and low-density lipoprotein receptor, as well as the protein level of 5′ adenosine monophosphate-activated protein kinase in the liver. The results clearly demonstrate that HVC1 has a potent hypolipidemic effect, and suggest that HVC1 should be evaluated as a potential treatment for hyperlipidemia. PMID:27510839

  15. Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in SH-SY5Y cells.

    PubMed

    Rosati, Fabiana; Sturli, Niccolò; Cungi, Maria Chiara; Morello, Matteo; Villanelli, Fabio; Bartolucci, Gianluca; Finocchi, Claudia; Peri, Alessandro; Serio, Mario; Danza, Giovanna

    2011-04-01

    Neurosteroids are involved in Central Nervous System development, brain functionality and neuroprotection but little is known about regulators of their biosynthesis. Recently gonadotropins, Gonadotropin-releasing Hormone (GnRH) and their receptors have been localized in different brain regions, such as hippocampus and cortex. Using human neuronal-like cells we found that GnRH up-regulates the expression of key genes of cholesterol and steroid synthesis when used in a narrow range around 1.0 nM. The expression of Hydroxysterol D24-reductase (seladin-1/DHCR24), that catalyzes the last step of cholesterol biosynthesis, is increased by 50% after 90 min of incubation with GnRH. StAR protein and P450 side chain cleavage (P450scc) are up-regulated by 3.3 times after 90 min and by 3.5 times after 3 h, respectively. GnRH action is mediated by LH and 1.0 nM GnRH enhances the expression of LHβ as well. A two fold increase of cell cholesterol is induced after 90 min of GnRH incubation and 17β-estradiol (E2) production is increased after 24, 48 and 72 h. These data indicate for the first time that GnRH regulates both cholesterol and steroid biosynthesis in human neuronal-like cells and suggest a new physiological role for GnRH in the brain. PMID:21296663

  16. Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models.

    PubMed

    Sirokmány, Gábor; Donkó, Ágnes; Geiszt, Miklós

    2016-04-01

    Nox/Duox NADPH oxidases are now considered the primary, regulated sources of reactive oxygen species (ROS). These enzymes are expressed in diverse cells and tissues, and their products are essential in several physiological settings. Knockout mouse models are instrumental in identifying the physiological functions of Nox/Duox enzymes as well as in exploring the impact of their pharmacological targeting on disease progression. The currently available data from experiments on knockout animals suggest that the lack of non-phagocytic Nox/Duox enzymes often modifies the course and phenotype in many disease models. Nevertheless, as illustrated by studies on Nox4-deficient animals, the absence of Nox-derived ROS can also lead to aggravated disease manifestation, reinforcing the need for a more balanced view on the role of ROS in health and disease.

  17. Amplified solid-state electrochemiluminescence detection of cholesterol in near-infrared range based on CdTe quantum dots decorated multiwalled carbon nanotubes@reduced graphene oxide nanoribbons.

    PubMed

    Huan, Juan; Liu, Qian; Fei, Airong; Qian, Jing; Dong, Xiaoya; Qiu, Baijing; Mao, Hanping; Wang, Kun

    2015-11-15

    An amplified solid-state electrochemiluminescence (ECL) biosensor for detection of cholesterol in near-infrared (NIR) range was constructed based on CdTe quantum dots (QDs) decorated multiwalled carbon nanotubes@reduced graphene nanoribbons (CdTe-MWCNTs@rGONRs), which were prepared by electrostatic interactions. The CdTe QDs decorated on the MWCNTs@rGONRs resulted in the amplified ECL intensity by ~4.5 fold and decreased onset potential by ~100 mV. By immobilization of the cholesterol oxidase (ChOx) and NIR CdTe-MWCNTs@rGONRs on the electrode surface, a solid-state ECL biosensor for cholesterol detection was constructed. When cholesterol was added to the detection solution, the immobilized ChOx catalyzed the oxidation of cholesterol to generate H2O2, which could be used as the co-reactant in the ECL system of CdTe-MWCNTs@rGONRs. The as-prepared biosensor exhibited good performance for cholesterol detection including good reproducibility, selectivity, and acceptable linear range from 1 μM to 1mM with a relative low detection limit of 0.33 μM (S/N=3). The biosensor was successfully applied to the determination of cholesterol in biological fluid and food sample, which would open a new possibility for development of solid-state ECL biosensors with NIR emitters.

  18. Physiological responses to daily light exposure

    PubMed Central

    Yang, Yefeng; Yu, Yonghua; Yang, Bo; Zhou, Hong; Pan, Jinming

    2016-01-01

    Long daylength artificial light exposure associates with disorders, and a potential physiological mechanism has been proposed. However, previous studies have examined no more than three artificial light treatments and limited metabolic parameters, which have been insufficient to demonstrate mechanical responses. Here, comprehensive physiological response curves were established and the physiological mechanism was strengthened. Chicks were illuminated for 12, 14, 16, 18, 20, or 22 h periods each day. A quadratic relationship between abdominal adipose weight (AAW) and light period suggested that long-term or short-term light exposure could decrease the amount of AAW. Quantitative relationships between physiological parameters and daily light period were also established in this study. The relationships between triglycerides (TG), cholesterol (TC), glucose (GLU), phosphorus (P) levels and daily light period could be described by quadratic regression models. TG levels, AAW, and BW positively correlated with each other, suggesting long-term light exposure significantly increased AAW by increasing TG thus resulting in greater BW. A positive correlation between blood triiodothyronine (T3) levels and BW suggested that daily long-term light exposure increased BW by thyroid hormone secretion. Though the molecular pathway remains unknown, these results suggest a comprehensive physiological mechanism through which light exposure affects growth. PMID:27098210

  19. Physiological responses to daily light exposure

    NASA Astrophysics Data System (ADS)

    Yang, Yefeng; Yu, Yonghua; Yang, Bo; Zhou, Hong; Pan, Jinming

    2016-04-01

    Long daylength artificial light exposure associates with disorders, and a potential physiological mechanism has been proposed. However, previous studies have examined no more than three artificial light treatments and limited metabolic parameters, which have been insufficient to demonstrate mechanical responses. Here, comprehensive physiological response curves were established and the physiological mechanism was strengthened. Chicks were illuminated for 12, 14, 16, 18, 20, or 22 h periods each day. A quadratic relationship between abdominal adipose weight (AAW) and light period suggested that long-term or short-term light exposure could decrease the amount of AAW. Quantitative relationships between physiological parameters and daily light period were also established in this study. The relationships between triglycerides (TG), cholesterol (TC), glucose (GLU), phosphorus (P) levels and daily light period could be described by quadratic regression models. TG levels, AAW, and BW positively correlated with each other, suggesting long-term light exposure significantly increased AAW by increasing TG thus resulting in greater BW. A positive correlation between blood triiodothyronine (T3) levels and BW suggested that daily long-term light exposure increased BW by thyroid hormone secretion. Though the molecular pathway remains unknown, these results suggest a comprehensive physiological mechanism through which light exposure affects growth.

  20. Serum cholesterol concentrations among Navajo Indians.

    PubMed

    Sugarman, J R; Gilbert, T J; Percy, C A; Peter, D G

    1992-01-01

    Navajo Indians have been reported by earlier investigators to have low concentrations of serum lipids and a low prevalence of hyperlipidemia, as well as low rates of ischemic heart disease. However, no data on serum lipid concentrations among Navajos have been reported for more than two decades. The authors conducted a study to determine the distribution of concentrations of serum total cholesterol (TC), high density lipoprotein cholesterol, low density lipoprotein cholesterol, and triglyceride among persons 25-74 years old living in a representative community on the Navajo Indian reservation. Data are reported for 255 subjects, 105 men and 150 women, ages 25-74 years. The authors compared these data to those for the general population as determined by the second National Health and Nutrition Examination Survey (NHANES II). TC concentrations among Navajo men were similar to those from NHANES II. TC concentrations among younger Navajo women were similar to those for women younger than 55 years from NHANES II, but were significantly lower among older Navajo women. While 27.6 percent of men ages 25-74 years studied in NHANES II had TC concentrations greater than 240 milligrams per deciliter, 33.8 percent of Navajo men had similarly elevated TC. However, the prevalence of serum TC concentrations greater than 240 milligrams per deciliter among Navajo women (17.5 percent) was about half that among women studied in NHANES II (32.9 percent). A similar pattern was found for low density lipoprotein cholesterol. The researchers concluded that Navajo Indians are no longer characterized by low serum lipid concentrations, that increased cholesterol concentrations may be a harbinger of increasing rates of atherosclerotic coronary heart disease among Navajos, and that attention should be directed to primary prevention of hyperlipidemia in Navajo Indian communities. PMID:1738814

  1. Role of Cholesterol Pathways in Norovirus Replication▿

    PubMed Central

    Chang, Kyeong-Ok

    2009-01-01

    Norwalk virus (NV) is a prototype strain of the noroviruses (family Caliciviridae) that have emerged as major causes of acute gastroenteritis worldwide. I have developed NV replicon systems using reporter proteins such as a neomycin-resistant protein (NV replicon-bearing cells) and a green fluorescent protein (pNV-GFP) and demonstrated that these systems were excellent tools to study virus replication in cell culture. In the present study, I first performed DNA microarray analysis of the replicon-bearing cells to identify cellular factors associated with NV replication. The analysis demonstrated that genes in lipid (cholesterol) or carbohydrate metabolic pathways were significantly (P < 0.001) changed by the gene ontology analysis. Among genes in the cholesterol pathways, I found that mRNA levels of hydroxymethylglutaryl-coenzyme A (HMG-CoA) synthase, squalene epoxidase, and acyl-CoA:cholesterol acyltransferase (ACAT), ACAT2, small heterodimer partner, and low-density lipoprotein receptor (LDLR)-related proteins were significantly changed in the cells. I also found that the inhibition of cholesterol biosynthesis using statins (an HMG-CoA reductase inhibitor) significantly increased the levels of NV proteins and RNA, whereas inhibitors of ACAT significantly reduced the replication of NV in replicon-bearing cells. Up- or downregulation of virus replication with these agents significantly correlated with the mRNA level of LDLR in replicon-bearing cells. Finally, I found that the expression of LDLR promoted NV replication in trans by transfection study with pNV-GFP. I conclude that the cholesterol pathways such as LDLR expression and ACAT activity may be crucial in the replication of noroviruses in cells, which may provide potential therapeutic targets for viral infection. PMID:19515767

  2. Mitochondrial terminal alternative oxidase and its enhancement by thermal stress in the coral symbiont Symbiodinium

    NASA Astrophysics Data System (ADS)

    Oakley, Clinton A.; Hopkinson, Brian M.; Schmidt, Gregory W.

    2014-06-01

    A terminal electron acceptor alternative to mitochondrial cytochrome c oxidase (COX), mitochondrial alternative oxidase (AOX), is ubiquitous in higher plants and represented in nearly every algal taxon but is poorly documented in dinoflagellates. AOX competes for electrons with the conventional COX and has been hypothesized to function as a means of reducing oxidative stress in mitochondria, as well as a potential mechanism for ameliorating thermal and other physiological stressors. Here, the presence of an active AOX in cultured Symbiodinium was assayed by the response of oxygen consumption to the AOX inhibitor salicylhydroxamic acid (SHAM) and the COX inhibitor cyanide (CN). CN-insensitive, SHAM-sensitive oxygen consumption was found to account for a large portion (26 %) of Symbiodinium dark respiration and is consistent with high levels of AOX activity. This experimental evidence of the existence of a previously unreported terminal oxidase was further corroborated by analysis of publicly available Symbiodinium transcriptome data. The potential for enhanced AOX expression to play a compensatory role in mediating thermal stress was supported by inhibitor assays of cultured Symbiodinium at low (18 °C), moderate (26 °C), and high (32 °C) temperature conditions. Maximum capacity of the putative AOX pathway as a proportion of total dark oxygen consumption was found to increase from 26 % at 26 °C to 45 % and 53 % at 18 °C and 32 °C, respectively, when cells were acclimated to the treatment temperatures. Cells assayed at 18 and 32 °C without acclimation exhibited either the same or lower AOX capacity as controls, suggesting that the AOX protein is upregulated under temperature stress. The physiological implications for the presence of AOX in the coral/algal symbiosis and its potential role in response to many forms of biotic and abiotic stress, particularly oxidative stress, are discussed.

  3. To Your Health: NLM update transcript - Cholesterol screening for kids?

    MedlinePlus

    ... transcript102416.html To Your Health: NLM update Transcript Cholesterol screening for kids? : 10/24/2016 To use ... the need for children to be screened for cholesterol levels in their bloodstream, finds a recent article ...

  4. Talk with Your Health Care Provider about High Cholesterol

    MedlinePlus

    ... you do? Always ask your provider what your cholesterol numbers are and write them down. Discuss these ... provider may prescribe medicine to help lower your cholesterol. y y Take your medicine every day, or ...

  5. Biliary cholesterol transport and precipitation: introduction and overview of conference.

    PubMed

    Strasberg, S M; Harvey, P R

    1990-09-01

    Cholesterol is secreted into bile as cholesterol-phospholipid vesicles. The cholesterol and phospholipid are subsequently exposed to the bile salts contained in the bile, which leads to the process of micellation. Two situations may arise depending on whether there is enough bile salt in proportion to cholesterol to complete this "maturation" process. If the cholesterol saturation is low, at equilibrium the bile salts will have completely micellized the vesicles. On the other hand, if bile is saturated with cholesterol, the micellation process is incomplete and vesicles and micelles will be present at equilibrium. The residual vesicle in this latter situation may have a higher cholesterol/phospholipid ratio because of the greater propensity of phospholipid to be micellized. This situation may result in cholesterol nucleation. The mechanism of nucleation from vesicles and the possible role of nucleating and antinucleating proteins in this process have been discussed.

  6. The process of cholesterol cholelithiasis induced by diet in the prairie dog: a physicochemical characterization.

    PubMed

    Holzbach, R T; Corbusier, C; Marsh, M; Naito, H K

    1976-06-01

    The rapid induction of cholesterol cholelithiasis in a new experimental model, the prairie dog, has recently been reported by two groups. In this model they were able to induce gallstones in as brief a period as 2 weeks, using a 1.2 per cent cholesterol diet. This unprecedented time intensity or telescoping of the induction process provided a unique opportunity for observation of physicochemical changes occurring rapidly over a short period of time and to correlate these with degree of biliary cholesterol saturation. To make such observations, 97 adult male and female prairie dogs were used in the present study. Seventy-two were fed the high cholesterol diet and sacrificed at intervals over a 14-day period; the remaining 25 were used as controls. The primary objective of this work was to determine whether or not there was any relationship between the in vivo events induced in prairie dog bile and our recently reported detailed observations of cholesterol precipitation phenomena in synthetic bile analogs. In these studies, solutions of physiologically relevant composition were constructed, which, when plotted according to convention on a tri-linear graph, fell within the zone of metastable or suspended supersaturation. These solutions revealed a consistent and previously undescribed liquid crystal to solid crystal phase transition during their approach to equilibrium at 37 degrees C. The in vivo studies of prairie dog bile following rapid induction of supersaturation revealed identical changes. At first, the supersaturated biles were isotropic followed by a period of turbidity and invariable formation of mesophase. After a few days, the liquid crystals decreased and solid cholesterol crystallites appeared. The most striking aspect of these observations is that the in vitro work has now predicted not only static, but dynamic processes as well, with respect to cholesterol precipitation beginning with the metastably supersaturated state and eventuating in gallstone

  7. Model parameters for simulation of physiological lipids

    PubMed Central

    McGlinchey, Nicholas

    2016-01-01

    Coarse grain simulation of proteins in their physiological membrane environment can offer insight across timescales, but requires a comprehensive force field. Parameters are explored for multicomponent bilayers composed of unsaturated lipids DOPC and DOPE, mixed‐chain saturation POPC and POPE, and anionic lipids found in bacteria: POPG and cardiolipin. A nonbond representation obtained from multiscale force matching is adapted for these lipids and combined with an improved bonding description of cholesterol. Equilibrating the area per lipid yields robust bilayer simulations and properties for common lipid mixtures with the exception of pure DOPE, which has a known tendency to form nonlamellar phase. The models maintain consistency with an existing lipid–protein interaction model, making the force field of general utility for studying membrane proteins in physiologically representative bilayers. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. PMID:26864972

  8. Endothelins and NADPH oxidases in the cardiovascular system.

    PubMed

    Dammanahalli, Karigowda J; Sun, Zhongjie

    2008-01-01

    1. The endothelin (ET) system and NADPH oxidase play important roles in the regulation of cardiovascular function, as well as in the pathogenesis of hypertension and other cardiovascular diseases. 2. Endothelins activate NADPH oxidases and thereby increase superoxide production, resulting in oxidative stress and cardiovascular dysfunction. Thus, NADPH oxidases may mediate the role of endothelins in some cardiovascular diseases. However, the role of reactive oxygen species (ROS) in mediating ET-induced vasoconstriction and cardiovascular disease remains under debate, as evidenced by conflicting reports from different research teams. Conversely, activation of NADPH oxidase can stimulate ET secretion via ROS generation, which further enhances the cardiovascular effects of NADPH oxidase. However, little is known about how ROS activate the endothelin system. It seems that the relationship between ET-1 and ROS may vary with cardiovascular disorders. 3. Endothelins activate NADPH oxidase via the ET receptor-proline-rich tyrosine kinase-2 (Pyk2)-Rac1 pathway. Rac1 is an important regulator of NADPH oxidase. There is ample evidence supporting direct stimulation by Rac1 of NADPH oxidase activity. In addition, Rac1-induced cardiomyocyte hypertrophy is mediated by the generation of ROS.

  9. Ezetimibe and Simvastatin Reduce Cholesterol Levels in Zebrafish Larvae Fed a High-Cholesterol Diet

    PubMed Central

    Baek, Ji Sun; Fang, Longhou; Li, Andrew C.; Miller, Yury I.

    2012-01-01

    Cholesterol-fed zebrafish is an emerging animal model to study metabolic, oxidative, and inflammatory vascular processes relevant to pathogenesis of human atherosclerosis. Zebrafish fed a high-cholesterol diet (HCD) develop hypercholesterolemia and are characterized by profound lipoprotein oxidation and vascular lipid accumulation. Using optically translucent zebrafish larvae has the advantage of monitoring vascular pathology and assessing the efficacy of drug candidates in live animals. Thus, we investigated whether simvastatin and ezetimibe, the principal drugs used in management of hypercholesterolemia in humans, would also reduce cholesterol levels in HCD-fed zebrafish larvae. We found that ezetimibe was well tolerated by zebrafish and effectively reduced cholesterol levels in HCD-fed larvae. In contrast, simvastatin added to water was poorly tolerated by zebrafish larvae and, when added to food, had little effect on cholesterol levels in HCD-fed larvae. Combination of low doses of ezetimibe and simvastatin had an additive effect in reducing cholesterol levels in zebrafish. These results suggest that ezetimibe exerts in zebrafish a therapeutic effect similar to that in humans and that the hypercholesterolemic zebrafish can be used as a low-cost and informative model for testing new drug candidates and for investigating mechanisms of action for existing drugs targeting dyslipidemia. PMID:22693663

  10. Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Fielding, P.E.; Miida, Takashi; Fielding, C.J. )

    1991-09-03

    The metabolism of cholesterol derived from ({sup 3}H) cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large {alpha}-migrating HDL (HDL{sub 2}) and was then transferred to small {alpha}-HDL (HDL{sub 3}) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating HDL{sub 2}. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL{sub 2} as a relatively unreactive sink for LCAT-derived cholesteryl esters.

  11. Characterization of ascorbate oxidase from Acremonium sp. HI-25.

    PubMed

    Hirose, J; Sakurai, T; Imamura, K; Watanabe, H; Iwamoto, H; Hiromi, K; Itoh, H; Shin, T; Murao, S

    1994-05-01

    The ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing four copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2. The EPR parameters of the type 1 and 2 copper atoms in the ascorbate oxidase are very similar to those in the case of the ascorbate oxidase obtained from cucumber, which is a dimeric protein. The apparent Km and kcat values for ascorbic acid of the ascorbate oxidase from Acremonium sp. HI-25 are almost the same as those of the monomeric unit of the ascorbate oxidase from cucumber. PMID:7961590

  12. 25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

    SciTech Connect

    Olsen, Brett N.; Schlesinger, Paul H.; Ory, Daniel S.; Baker, Nathan A.

    2011-02-01

    Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work, using molecular dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers to examine interactions between 25-HC and cholesterol in the same bilayer. When added to cholesterol-containing membranes, 25-HC causes larger changes in membrane structure than when added to cholesterol-free membranes, demonstrating interactions between the two sterols. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and therefore its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These interactions provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through direct modulation of cholesterol availability.

  13. Carbon Inverse Opal Rods for Nonenzymatic Cholesterol Detection.

    PubMed

    Zhong, Qifeng; Xie, Zhuoying; Ding, Haibo; Zhu, Cun; Yang, Zixue; Gu, Zhongze

    2015-11-18

    Carbon inverse opal rods made from silica photonic crystal rods are used for nonenzymatic cholesterol sensing. The characteristic reflection peak originating from the physical periodic structure works as sensing signals for quantitatively estimating cholesterol concentrations. Carbon inverse opal rods work both in cholesterol standard solutions and human serum. They are suitable for practical use in clinical diagnose. PMID:26415111

  14. Understanding Lipoproteins as Transporters of Cholesterol and Other Lipids

    ERIC Educational Resources Information Center

    Biggerstaff, Kyle D.; Wooten, Joshua S.

    2004-01-01

    A clear picture of lipoprotein metabolism is essential for understanding the pathophysiology of atherosclerosis. Many students are taught that low-density lipoprotein-cholesterol is "bad" and high-density lipoprotein-cholesterol is "good." This misconception leads to students thinking that lipoproteins are types of cholesterol rather than…

  15. Carbon Inverse Opal Rods for Nonenzymatic Cholesterol Detection.

    PubMed

    Zhong, Qifeng; Xie, Zhuoying; Ding, Haibo; Zhu, Cun; Yang, Zixue; Gu, Zhongze

    2015-11-18

    Carbon inverse opal rods made from silica photonic crystal rods are used for nonenzymatic cholesterol sensing. The characteristic reflection peak originating from the physical periodic structure works as sensing signals for quantitatively estimating cholesterol concentrations. Carbon inverse opal rods work both in cholesterol standard solutions and human serum. They are suitable for practical use in clinical diagnose.

  16. Dietary cholesterol and the plasma lipids and lipoproteins in the Tarahumara Indians: a people habituated to a low cholesterol diet after weaning.

    PubMed

    McMurry, M P; Connor, W E; Cerqueira, M T

    1982-04-01

    Eight Tarahumara Indian men participated in a metabolic study to measure the responsiveness of their plasma cholesterol levels to dietary cholesterol. They were fed isocaloric cholesterol-free and high cholesterol diets containing 20% fat, 15% protein, and 65% carbohydrate calories. On admission to the study, the Tarahumaras had a low mean plasma cholesterol concentration (120 mg/dl), reflecting their habitual low cholesterol diet. After 3 wk of a cholesterol-free diet their cholesterol levels were 113 mg/dl. The men were then fed a high cholesterol diet (1000 mg/day) which increased the mean total plasma cholesterol to 147 mg/dl (p less than 0.01) and also increased the low-density lipoprotein cholesterol concentration. Tarahumaras, habituated to a low cholesterol diet after weaning, had the typical hypercholesterolemic response to a high cholesterol diet that has been previously observed in subjects whose lifelong diet was high in cholesterol content.

  17. Effects of Cholesterol-altering Pharmaceuticals on Cholesterol Metabolism, Steroidogenesis, and Gene Expression in the Fathead Minnow (Pimephales promelas)

    EPA Science Inventory

    Pharmaceuticals that target cholesterol biosynthesis and uptake are among the most widely prescribed drugs and have been detected in the aquatic environment. Fibrates are a class of pharmaceuticals that indirectly modulate cholesterol biosynthesis through effects on peroxisome pr...

  18. The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin.

    PubMed

    Kim, Young-Mi; Kim, Eun-Cheol; Kim, Youngho

    2011-01-01

    The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.

  19. Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties.

    PubMed

    de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Voisin, Maud; Mhamdi, Loubna; Dalenc, Florence; Lacroix-Triki, Magali; Filleron, Thomas; Pont, Frederic; Saati, Talal Al; Morisseau, Christophe; Hammock, Bruce D; Silvente-Poirot, Sandrine; Poirot, Marc

    2013-01-01

    We previously synthesized dendrogenin A and hypothesized that it could be a natural metabolite occurring in mammals. Here we explore this hypothesis and report the discovery of dendrogenin A in mammalian tissues and normal cells as an enzymatic product of the conjugation of 5,6α-epoxy-cholesterol and histamine. Dendrogenin A was not detected in cancer cell lines and was fivefold lower in human breast tumours compared with normal tissues, suggesting a deregulation of dendrogenin A metabolism during carcinogenesis. We established that dendrogenin A is a selective inhibitor of cholesterol epoxide hydrolase and it triggered tumour re-differentiation and growth control in mice and improved animal survival. The properties of dendrogenin A and its decreased level in tumours suggest a physiological function in maintaining cell integrity and differentiation. The discovery of dendrogenin A reveals a new metabolic pathway at the crossroads of cholesterol and histamine metabolism and the existence of steroidal alkaloids in mammals.

  20. Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties

    PubMed Central

    de Medina, Philippe; Paillasse, Michael R.; Segala, Gregory; Voisin, Maud; Mhamdi, Loubna; Dalenc, Florence; Lacroix-Triki, Magali; Filleron, Thomas; Pont, Frederic; Saati, Talal Al; Morisseau, Christophe; Hammock, Bruce D.; Silvente-Poirot, Sandrine; Poirot, Marc

    2013-01-01

    We previously synthesized dendrogenin A and hypothesized that it could be a natural metabolite occurring in mammals. Here we explore this hypothesis and report the discovery of dendrogenin A in mammalian tissues and normal cells as an enzymatic product of the conjugation of 5,6α-epoxy-cholesterol and histamine. Dendrogenin A was not detected in cancer cell lines and was fivefold lower in human breast tumours compared with normal tissues, suggesting a deregulation of dendrogenin A metabolism during carcinogenesis. We established that dendrogenin A is a selective inhibitor of cholesterol epoxide hydrolase and it triggered tumour re-differentiation and growth control in mice and improved animal survival. The properties of dendrogenin A and its decreased level in tumours suggest a physiological function in maintaining cell integrity and differentiation. The discovery of dendrogenin A reveals a new metabolic pathway at the crossroads of cholesterol and histamine metabolism and the existence of steroidal alkaloids in mammals. PMID:23673625

  1. Understanding Cholesterol and Heart Health | NIH MedlinePlus the Magazine

    MedlinePlus

    ... cholesterol throughout the body: Low-density lipoproteins (LDL): LDL cholesterol sometimes is called "bad" cholesterol. A high LDL ... or even death. The higher the level of LDL cholesterol in your blood, the GREATER your chance is ...

  2. Effects of statins and cholesterol on memory functions in mice.

    PubMed

    Ghodke, Ravindra M; Tour, Nagesh; Devi, Kshama

    2012-12-01

    Studies on influence of lipid lowering therapies have generated wide controversial results on the role of cholesterol on memory function. However recent studies revealed that cholesterol lowering treatment substantially reduce the risk of dementia. The objectives of this study were to analyze the effect of statins on memory function and to establish the relationship between increase/decrease in cholesterol synthesis, total cholesterol level and memory function in animals. We examined the relationship between biosynthesis of cholesterol and memory function using two statins (lipophilic simvastatin and hydrophilic pravastatin) and high cholesterol diet in mice for 15 days and 4 months. Memory performance was evaluated with two different behavioral tests and various biochemical parameters such as serum cholesterol, whole brain cholesterol, brain 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) activity and brain acetylcholine esterase (AChE) activity. We found that statin treatment for 4 months, but not for 15 days, showed significant improvement in memory function whereas high cholesterol diet showed significant impairment of memory. However long-term statin treatment showed significant decrease in serum cholesterol level as well as brain AChE level. Moreover high cholesterol diet showed significant decrease in memory function with an increase in serum cholesterol level as well as brain AChE level. There is no direct correlation between brain cholesterol level, as well as HMG-CoA activity with memory function regulation. However there is definite link between plasma cholesterol level and AChE level. A long-standing plasma cholesterol alteration may be essential to regulate memory function which in turn might be mediated through AChE modulated pathway.

  3. Effects of saturated and unsaturated fats given with and without dietary cholesterol on hepatic cholesterol synthesis and hepatic lipid metabolism.

    PubMed

    Bochenek, W; Rodgers, J B

    1978-01-27

    Hepatic cholesterol synthesis was studied in rats after consuming diets of varying neutral lipid and cholesterol content. Cholesterol synthesis was evaluated by measuring 3-hydroxy-3-methylglutaryl-CoA reductase and by determining the rate of 3H-labeled sterol production from [3H]mevalonate. Results were correlated with sterol balance data and hepatic lipid content. Hepatic cholesterol synthesis was relatively great when cholesterol was excluded from the diet. The source of neutral dietary lipids, saturated vs. unsaturated, produced no change in hepatic sterol synthesis. Values for fecal sterol outputs and hepatic cholesterol levels were also similar in rats consuming either saturated or unsaturated fats. When 1% cholesterol was added to the diet, hepatic cholesterol synthesis was suppressed but the degree of suppression was greater in rats consuming unsaturated vs. saturated fats. This was associated with greater accumulation of cholesterol in livers from rats consuming unsaturates and a reduction in fecal neutral sterol output in this group as opposed to results from rats on saturated fats. Cholesterol consumption also altered the fatty acid composition of hepatic phospholipids producing decreases in the percentages of essential polyunsaturated fatty acids. It is concluded that dietary cholesterol alters cholesterol and fatty acid metabolism in the liver and that this effect is enhanced by dietary unsaturated fats.

  4. Walking and serum cholesterol in adults.

    PubMed Central

    Tucker, L A; Friedman, G M

    1990-01-01

    We measured the association between walking for exercise and the ratio of total cholesterol/HDL cholesterol in 3,621 adults. After controlling for age, gender, income, body fat, alcohol use, exercise other than walking, and cigarette smoking, adults in the high, moderate, and low duration walking categories were compared to those in the no walking-no exercise category. The relative risk for total/HDL ratios of 5.0 or more were .46 (95% CI = .27, .80), .48 (95% total/HDL ratios of 5.0 or more were .46 (95% CI = .27, .80), .48 (95% CI = .30, .76), and 1.11 (95% CI = .81, 1.53) respectively. PMID:2382750

  5. Lipid rafts, cholesterol, and the brain

    PubMed Central

    Korade, Zeljka; Kenworthy, Anne K.

    2008-01-01

    Summary Lipid rafts are specialized membrane microdomains that serve as organizing centers for assembly of signaling molecules, influence membrane fluidity and trafficking of membrane proteins, and regulate different cellular processes such as neurotransmission and receptor trafficking. In this article, we provide an overview of current methods for studying lipid rafts and models for how lipid rafts might form and function. Next, we propose a potential mechanism for regulating lipid rafts in the brain via local control of cholesterol biosynthesis by neurotrophins and their receptors. Finally, we discuss evidence that altered cholesterol metabolism and/or lipid rafts play a critical role in the pathophysiology of multiple CNS disorders, including Smith-Lemli-Opitz syndrome, Huntington, Alzheimer's, and Niemman-Pick Type C diseases. PMID:18402986

  6. Cholesterol efflux capacity: An introduction for clinicians.

    PubMed

    Anastasius, Malcolm; Kockx, Maaike; Jessup, Wendy; Sullivan, David; Rye, Kerry-Anne; Kritharides, Leonard

    2016-10-01

    Epidemiologic studies have shown an inverse correlation between high-density lipoprotein (HDL) cholesterol (HDL-C) levels and cardiovascular disease outcomes. However, the hypothesis of a causal relationship between HDL-C and cardiovascular disease has been challenged by genetic and clinical studies. Serum cholesterol efflux capacity (CEC) is an important measure of HDL function in humans. Recent large clinical studies have shown a correlation between in vitro CEC and cardiovascular disease prevalence and incidence, which appears to be independent of HDL-C concentration. The present review summarizes recent large clinical studies and introduces important methodological considerations. Further studies are required to standardize and establish the reproducibility of this measure of HDL function and clarify whether modulating CEC will emerge as a useful therapeutic target. PMID:27659883

  7. Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea.

    PubMed

    McDonald, Allison E; Vanlerberghe, Greg C

    2005-04-11

    Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.

  8. Rapid cholesterol nucleation time and cholesterol gall stone formation after subtotal or total colectomy in humans.

    PubMed Central

    Makino, I; Chijiiwa, K; Higashijima, H; Nakahara, S; Kishinaka, M; Kuroki, S; Mibu, R

    1994-01-01

    Changes in biliary lipid composition, pH, ionised calcium, total and unconjugated bilirubin, and cholesterol nucleation time of gall bladder bile samples were examined in six patients who had undergone subtotal or total colectomy between five months and seven years previously, and values were compared with those in control patients with no gall stones. The colectomy group mainly comprised patients with ulcerative colitis and familial adenomatosis coli, in whom only a short length of the terminal ileum (mean (SEM) 2.25 (0.57) cm) had been resected. The reconstruction procedures were ileoanal anastomosis in two patients, terminal ileostomy in two, ileorectal anastomosis in one, and J shaped ileal pouch-anal anastomosis in one patient. The distributions of age, sex, and relative body weight were similar in the two groups. The gall bladder bile was lithogenic in the post colectomy group--these patients had a significantly increased cholesterol saturation index (p < 0.01) and rapid cholesterol nucleation time (p < 0.05) compared with the control group. A significant increase in the molar percentage of cholesterol and a decrease in that of total bile acid associated with significantly decreased secondary bile acids (p < 0.05) were observed in the post colectomy group. Gall stones formed in two of six patients after colectomy were cholesterol stones containing more than 80% cholesterol by dry weight. Total and unconjugated bilirubin, pH, and ionised calcium values were similar in the two groups. The results indicate that after total or subtotal colectomy the composition of gall bladder bile increases the risk of cholesterol gall stone formation. PMID:7829016

  9. PCSK9 and LDLR The Yin-Yang in the Cellular Uptake of Cholesterol.

    PubMed

    Morales-Villegas, E

    2014-07-01

    The intracellular concentration of cholesterol is a vital constant influenced by the uptake, metabolism and excretion of cholesterol. The synthesis and expression of the PCSK9-LDLR duo is one of the most important mechanisms to regulate this constant; in a physiological state, the yin-yang balance between PCSK9 and LDLR regulates the entry of cholesterol into the cell to keep the intracellular cholesterol concentration stable. The mapping of the human gene encoding the serine protease PCSK9, located at 1p32-3, has allowed the identification of mutations with "gain" and "loss" of protease functions. Gain of function mutations causes decreased LDLR resulting in increased LDL-C and increased incidence of cardiovascular events. Loss of function mutations have opposite effect, increased LDLR, decreased LDL-C and decreased incidence of cardiovascular events. The identification of human mutations with PCSK9 "loss" of function demostrated the benefit of decreased PCSK9 and opened the door to developing new anti-PCSK9 therapies. The goal of this research area is to reduce the incidence of cardiovascular events beyond statins; the strategy is to mimic the state of PCSK9 "loss" of function by tactics as oligonucleotide therapies targeting PCSK9 mRNA and/or biological therapies with human monoclonal antibodies targeting PCSK9. This chapter reviews, the characteristics of the PCSK9, the physiological significance of the PCSK9-LDLR duo, and the therapeutic implications of the human genetic models of PCSK9 "loss" of function. The phase I-II clinical trial data of two promising monoclonal antibodies to PCSK9, Alirocumab formerly SAR236553/REGN727and AMG145 will be presented. PMID:24993279

  10. Mitotic spindle defects and chromosome mis-segregation induced by LDL/cholesterol-implications for Niemann-Pick C1, Alzheimer's disease, and atherosclerosis.

    PubMed

    Granic, Antoneta; Potter, Huntington

    2013-01-01

    consumption reduces the risk of developing atherosclerosis or AD. These results suggest a novel, cell cycle mechanism by which aberrant cholesterol homeostasis promotes neurodegeneration and atherosclerosis by disrupting chromosome segregation and potentially other aspects of microtubule physiology.

  11. Cholesterol in serum lipoprotein fractions after spaceflight

    NASA Technical Reports Server (NTRS)

    Leach, Carolyn S.; Johnson, Philip C., Jr.; Krauhs, Jane M.; Cintron, Nitza M.

    1988-01-01

    Results are reported from blood-lipid measurements obtained from 125 Space Shuttle crew members before and after space flight. The data are presented in tables and discussed in detail. The main differences noted between preflight and postflight values are a 12.8-percent decrease in high-density lipoproteins on postflight day 1 and significant decreases in total cholesterol and both high- and low-density lipoproteins later in the 23-day postflight period.

  12. Potent and selective mediators of cholesterol efflux

    DOEpatents

    Bielicki, John K; Johansson, Jan

    2015-03-24

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  13. Cholesterol - Multiple Languages: MedlinePlus

    MedlinePlus

    ... 繁體中文) French (français) Hindi (हिन्दी) Japanese (日本語) Korean (한국어) Portuguese (português) Russian (Русский) Somali (af Soomaali) ... コレステロール - 日本語 (Japanese) Bilingual PDF Health Information Translations Korean (한국어) Cholesterol 콜레스테롤 - 한국어 (Korean) Bilingual PDF Health ...

  14. Inhibition of cholesterol biosynthesis by Delta22-unsaturated phytosterols via competitive inhibition of sterol Delta24-reductase in mammalian cells.

    PubMed Central

    Fernández, Carlos; Suárez, Yajaira; Ferruelo, Antonio J; Gómez-Coronado, Diego; Lasunción, Miguel A

    2002-01-01

    Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [(14)C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Delta(24)-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (beta-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol (24)-reductase was confirmed in rat liver microsomes by using (14)C-labelled desmosterol as the substrate. The (22)-unsaturated phytosterols acted as competitive inhibitors of sterol (24)-reductase, with K(i) values (41.1, 42.7 and 36.8 microM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated K(m) for desmosterol (26.3 microM). The sterol 5,22-cholestedien-3beta-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of (24)-reductase (K(i)=3.34 microM). The usually low intracellular concentrations of the physiological substrates of (24)-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol (24)-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain (22)-unsaturated sterols as effective hypocholesterolaemic

  15. Response of the Cholesterol Metabolism to a Negative Energy Balance in Dairy Cows Depends on the Lactational Stage

    PubMed Central

    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. PMID:26034989

  16. Cholesterol gallstone formation in overweight mice establishes that obesity per se is not linked directly to cholelithiasis risk.

    PubMed

    Bouchard, Guylaine; Johnson, Derek; Carver, Tonya; Paigen, Beverly; Carey, Martin C

    2002-07-01

    The relationship between obesity and cholesterol cholelithiasis is not well understood at physiologic or genetic levels. To clarify whether obesity per se leads to increased prevalence of cholelithiasis, we examined cholesterol gallstone susceptibility in three polygenic (KK/H1J, NON/LtJ, NOD/LtJ) and five monogenic [carboxypeptidase E (Cpe (fat)), agouti yellow (A(y)), tubby (tub), leptin (Lep(ob)), leptin receptor (Lepr (db))] murine models of obesity during ingestion of a lithogenic diet containing dairy fat, cholesterol, and cholic acid. At 8 weeks on the diet, one strain of polygenic obese mice was resistant whereas the others revealed low or intermediate prevalence rates of cholelithiasis. Monogenic obese mice showed distinct patterns with either high or low gallstone prevalence rates depending upon the mutation. Dysfunction of the leptin axis, as evidenced by the Lep(ob) and the Lepr (db) mutations, markedly reduced gallstone formation in a genetically susceptible background strain, indicating that in mice with this genetic background, physiologic leptin homeostasis is a requisite for cholesterol cholelithogenesis. In contrast, the Cpe (fat) mutation enhanced the prevalence of cholelithiasis markedly when compared with the background strain. Since CPE converts many prohormones to hormones, a deficiency of biologically active cholecystokinin is a likely contributor to enhanced susceptibility to cholelithiasis through compromising gallbladder contractility and small intestinal motility. Because some murine models of obesity increased, whereas others decreased cholesterol gallstone susceptibility, we establish that cholesterol cholelithiasis in mice is not simply a secondary consequence of obesity per se. Rather, specific genes and distinct pathophysiological pathways are responsible for the shared susceptibility to both of these common diseases.

  17. 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. PMID:26034989

  18. Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

    PubMed Central

    Daou, Marianne; Piumi, François; Cullen, Daniel; Record, Eric

    2016-01-01

    ABSTRACT The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these

  19. Positive effect of dietary lutein and cholesterol on the undirected song activity of an opportunistic breeder

    PubMed Central

    Pinxten, Rianne; Zaid, Erika; Eens, Marcel

    2016-01-01

    Song is a sexually selected trait that is thought to be an honest signal of the health condition of an individual in many bird species. For species that breed opportunistically, the quantity of food may be a determinant of singing activity. However, it is not yet known whether the quality of food plays an important role in this respect. The aim of the present study was to experimentally investigate the role of two calorie-free nutrients (lutein and cholesterol) in determining the expression of a sexually selected behavior (song rate) and other behaviors (locomotor activity, self-maintenance activity, eating and resting) in male zebra finches (Taeniopygia guttata). We predicted that males supplemented with lutein and cholesterol would sing at higher rates than controls because both lutein and cholesterol have important health-related physiological functions in birds and birdsong mirrors individual condition. To control for testosterone secretion that may upregulate birdsong, birds were exposed to a decreasing photoperiod. Our results showed that control males down-regulated testosterone in response to a decreasing photoperiod, while birds treated with lutein or cholesterol maintained a constant singing activity. Both lutein- and cholesterol-supplemented groups sang more than control groups by the end of the experiment, indicating that the quality of food can affect undirected song irrespective of circulating testosterone concentrations. None of the other measured behaviors were affected by the treatment, suggesting that, when individuals have full availability of food, sexually selected song traits are more sensitive to the effect of food quality than other behavioral traits. Overall the results support our prediction that undirected song produced by male zebra finches signals access to high-quality food. PMID:27761321

  20. Enzymatic polymerization of dihydroquercetin using bilirubin oxidase.

    PubMed

    Khlupova, M E; Vasil'eva, I S; Shumakovich, G P; Morozova, O V; Chertkov, V A; Shestakova, A K; Kisin, A V; Yaropolov, A I

    2015-02-01

    Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and (1)H and (13)C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity.

  1. [NADPH oxidases, Nox: new isoenzymes family].

    PubMed

    Chuong Nguyen, Minh Vu; Lardy, Bernard; Paclet, Marie-Hélène; Rousset, Francis; Berthier, Sylvie; Baillet, Athan; Grange, Laurent; Gaudin, Philippe; Morel, Françoise

    2015-01-01

    NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.

  2. Degradation of pentachlorophenol by potato polyphenol oxidase.

    PubMed

    Hou, Mei-Fang; Tang, Xiao-Yan; Zhang, Wei-De; Liao, Lin; Wan, Hong-Fu

    2011-11-01

    In this study, polyphenol oxidase (PPO) was extracted from commercial potatoes. Degradation of pentachlorophenol by potato PPO was investigated. The experimental results show that potato PPO is more active in weak acid than in basic condition and that the optimum pH for the reaction is 5.0. The degradation of pentachlorophenol by potato PPO reaches a maximum at 298 K. After reaction for 1 h, the removal of both pentachlorophenol and total organic carbon is >70% with 6.0 units/mL potato PPO at pH 5.0 and 298 K. Pentachlorophenol can be degraded through dechlorination and ring-opening by potato PPO. The work demonstrates that pentachlorophenol can be effectively eliminated by crude potato PPO. PMID:21967325

  3. Visualization of monoamine oxidase in human brain

    SciTech Connect

    Fowler, J.S.; Volkow, N.D.; Wang, G.J.; Pappas, N.; Shea, C.; MacGregor, R.R.; Logan, J.

    1996-12-31

    Monoamine oxidase is a flavin enzyme which exists in two subtypes, MAO A and MAO B. In human brain MAO B predominates and is largely compartmentalized in cell bodies of serotonergic neurons and glia. Regional distribution of MAO B was determined by positron computed tomography with volunteers after the administration of deuterium substituted [11C]L-deprenyl. The basal ganglia and thalamus exhibited the greatest concentrations of MAO B with intermediate levels in the frontal cortex and cingulate gyrus while lowest levels were observed in the parietal and temporal cortices and cerebellum. We observed that brain MAO B increases with are in health normal subjects, however the increases were generally smaller than those revealed with post-mortem studies.

  4. Drugs related to monoamine oxidase activity.

    PubMed

    Fišar, Zdeněk

    2016-08-01

    Progress in understanding the role of monoamine neurotransmission in pathophysiology of neuropsychiatric disorders was made after the discovery of the mechanisms of action of psychoactive drugs, including monoamine oxidase (MAO) inhibitors. The increase in monoamine neurotransmitter availability, decrease in hydrogen peroxide production, and neuroprotective effects evoked by MAO inhibitors represent an important approach in the development of new drugs for the treatment of mental disorders and neurodegenerative diseases. New drugs are synthesized by acting as multitarget-directed ligands, with MAO, acetylcholinesterase, and iron chelation as targets. Basic information is summarized in this paper about the drug-induced regulation of monoaminergic systems in the brain, with a focus on MAO inhibition. Desirable effects of MAO inhibition include increased availability of monoamine neurotransmitters, decreased oxidative stress, decreased formation of neurotoxins, induction of pro-survival genes and antiapoptotic factors, and improved mitochondrial functions.

  5. ROS signalling, NADPH oxidases and cancer.

    PubMed

    Landry, William D; Cotter, Thomas G

    2014-08-01

    ROS (reactive oxygen species) have long been regarded as a series of destructive molecules that have a detrimental effect on cell homoeostasis. In support of this are the myriad antioxidant defence systems nearly all eukaryotic cells have that are designed to keep the levels of ROS in check. However, research data emerging over the last decade have demonstrated that ROS can influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules such as cAMP. Hydrogen peroxide (H2O2) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a wide range of cellular functions. Its localized synthesis by the Nox (NADPH oxidase) family of enzymes and how these enzymes are regulated is of particular interest to those who work in the field of tumour biology.

  6. Human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Jauhiainen, M.; Stevenson, K.J.; Dolphin, P.J.

    1988-05-15

    Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. The authors proposed a covalent catalytic mechanism of action for LCAT in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols they have probed the geometry of the catalytic site. They conclude that the two catalytic cysteine residues of LCAT (Cys/sup 31/ and Cys /sup 184/) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by teh bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

  7. HDL and cholesterol handling in the brain.

    PubMed

    Vitali, Cecilia; Wellington, Cheryl L; Calabresi, Laura

    2014-08-01

    Cholesterol is an essential component of both the peripheral nervous system and central nervous system (CNS) of mammals. Brain cholesterol is synthesized in situ by astrocytes and oligodendrocytes and is almost completely isolated from other pools of cholesterol in the body, but a small fraction can be taken up from the circulation as 27-hydroxycholesterol, or via the scavenger receptor class B type I. Glial cells synthesize native high-density lipoprotein (HDL)-like particles, which are remodelled by enzymes and lipid transfer proteins, presumably as it occurs in plasma. The major apolipoprotein constituent of HDL in the CNS is apolipoprotein E, which is produced by astrocytes and microglia. Apolipoprotein A-I, the major protein component of plasma HDL, is not synthesized in the CNS, but can enter and become a component of CNS lipoproteins. Low HDL-C levels have been shown to be associated with cognitive impairment and various neurodegenerative diseases. On the contrary, no clear association with brain disorders has been shown in genetic HDL defects, with the exception of Tangier disease. Mutations in a wide variety of lipid handling genes can result in human diseases, often with a neuronal phenotype caused by dysfunctional intracellular lipid trafficking.

  8. Neurosteroids: oligodendrocyte mitochondria convert cholesterol to pregnenolone

    SciTech Connect

    Hu, Z.Y.; Bourreau, E.; Jung-Testas, I.; Robel, P.; Baulieu, E.E.

    1987-12-01

    Oligodendrocyte mitochondria from 21-day-old Sprague-Dawley male rats were incubated with 100 nM (/sup 3/H)cholesterol. It yielded (/sup 3/H)pregnenolone at a rate of 2.5 +/- 0.7 and 5-(/sup 3/H)pregnene-3..beta..,20..cap alpha..-diol at a rate of 2.5 +/- 1.1 pmol per mg of protein per hr. Cultures of glial cells from 19- to 21-day-old fetuses (a mixed population of astrocytes and oligodendrocytes) were incubated for 24 hr with (/sup 3/H)mevalonolactone. (/sup 3/H)Cholesterol, (/sup 3/H)pregnenolone, and 5-(/sup 3/H)pregnene-3..beta..,20..cap alpha..-diol were characterized in cellular extracts. The formation of the /sup 3/H-labeled steroids was increased by dibutyryl cAMP (0.2 mM) added to the culture medium. The active cholesterol side-chain cleavage mechanism, recently suggested immunohistochemically and already observed in cultures of C6 glioma cells, reinforces the concept of neurosteroids applied to ..delta../sup 5/-3..beta..-hydroxysteroids previously isolated from brain.

  9. POLYAMINE OXIDASE 1 from rice (Oryza sativa) is a functional ortholog of Arabidopsis POLYAMINE OXIDASE 5

    PubMed Central

    Liu, Taibo; Wook Kim, Dong; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2014-01-01

    POLYAMINE OXIDASE 1 (OsPAO1), from rice (Oryza sativa), and POLYAMINE OXIDASE 5 (AtPAO5), from Arabidopsis (Arabidopsis thaliana), are enzymes sharing high identity at the amino acid level and with similar characteristics, such as polyamine specificity and pH preference; furthermore, both proteins localize to the cytosol. A loss-of-function Arabidopsis mutant, Atpao5–2, was hypersensitive to low doses of exogenous thermospermine but this phenotype could be rescued by introduction of the wild-type AtPAO5 gene. Introduction of OsPAO1, under the control of a constitutive promoter, into Atpao5–2 mutants also restored normal thermospermine sensitivity, allowing growth in the presence of low levels of thermospermine, along with a concomitant decrease in thermospermine content in plants. By contrast, introduction of OsPAO3, which encodes a peroxisome-localized polyamine oxidase, into Atpao5–2 plants could not rescue any of the mutant phenotypes in the presence of thermospermine. These results suggest that OsPAO1 is the functional ortholog of AtPAO5. PMID:25763711

  10. Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

    PubMed

    Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

    2016-10-01

    Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process. PMID:27295021

  11. Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

    PubMed Central

    Houille-Vernes, Laura; Rappaport, Fabrice; Wollman, Francis-André; Alric, Jean; Johnson, Xenie

    2011-01-01

    By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b6f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b6f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b6f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase. PMID:22143777

  12. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  13. Crystallization and preliminary X-ray diffraction analysis of the flax cytokinin oxidase LuCKX1.1

    PubMed Central

    Wan, Li; Williams, Simon J.; Zhang, Xiaoxiao; Ericsson, Daniel J.; Koeck, Markus; Dodds, Peter N.; Ellis, Jeffrey G.; Kobe, Bostjan

    2013-01-01

    The plant hormones cytokinins play a central role in regulating cell division and developmental events. Cytokinin oxidase regulates the levels of these plant hormones by catalyzing their irreversible oxidation, which contributes to the regulation of various morpho-physiological processes controlled by cytokinins. In this study, the crystallization and preliminary X-ray diffraction analysis of the flax cytokinin oxidase LuCKX1.1 are reported. Plate-like crystals of LuCKX1.1 were obtained using PEG 3350 as a precipitant and diffracted X-rays to 1.78 Å resolution. The protein crystals have the symmetry of space group C2 and are most likely to contain two molecules per asymmetric unit. PMID:24100555

  14. Physiological Information Database (PID)

    EPA Science Inventory

    EPA has developed a physiological information database (created using Microsoft ACCESS) intended to be used in PBPK modeling. The database contains physiological parameter values for humans from early childhood through senescence as well as similar data for laboratory animal spec...

  15. Dietary cholesterol, heart disease risk and cognitive dissonance.

    PubMed

    McNamara, Donald J

    2014-05-01

    In the 1960s, the thesis that dietary cholesterol contributes to blood cholesterol and heart disease risk was a rational conclusion based on the available science at that time. Fifty years later the research evidence no longer supports this hypothesis yet changing the dietary recommendation to limit dietary cholesterol has been a slow and at times contentious process. The preponderance of the clinical and epidemiological data accumulated since the original dietary cholesterol restrictions were formulated indicate that: (1) dietary cholesterol has a small effect on the plasma cholesterol levels with an increase in the cholesterol content of the LDL particle and an increase in HDL cholesterol, with little effect on the LDL:HDL ratio, a significant indicator of heart disease risk, and (2) the lack of a significant relationship between cholesterol intake and heart disease incidence reported from numerous epidemiological surveys. Over the last decade, many countries and health promotion groups have modified their dietary recommendations to reflect the current evidence and to address a now recognised negative consequence of ineffective dietary cholesterol restrictions (such as inadequate choline intake). In contrast, health promotion groups in some countries appear to suffer from cognitive dissonance and continue to promote an outdated and potentially hazardous dietary recommendation based on an invalidated hypothesis. This review evaluates the evidence for and against dietary cholesterol restrictions and the potential consequences of such restrictions.

  16. Effects of Charged Cholesterol Derivatives on Aβ40 Amyloid Formation.

    PubMed

    Elbassal, Esmail A; Liu, Haiyang; Morris, Clifford; Wojcikiewicz, Ewa P; Du, Deguo

    2016-01-14

    Understanding of the mechanistic progess of amyloid-β peptide (Aβ) aggregation is critical for elucidating the underlying pathogenesis of Alzheimer's disease (AD). Herein, we report for the first time the effects of two cholesterol derivatives, negatively charged cholesterol sulfate (cholesterol-SO4) and positively charged 3β-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol), on the fibrillization of Aβ40. Our results demonstrate that both of the nonvesicular forms of cholesterol-SO4 and DC-cholesterol moderately accelerate the aggregation rate of Aβ40. This effect is similar to that observed for unmodified cholesterol, indicating the importance of hydrophobic interactions in binding of Aβ40 to these steroid molecules. Furthermore, we show that the vesicles formed at higher concentrations of anionic cholesterol-SO4 facilitate Aβ40 aggregation rate markedly. In contrast, the cationic DC-cholesterol vesicles show the ability to inhibit Aβ40 fibril formation under appropriate experimental conditions. The results suggest that the electrostatic interactions between Aβ40 and the charged vesicles can be of great importance in regulating Aβ40-vesicle interaction. Our results also indicate that the structural properties of the aggregates of the cholesterol derivatives, including the surface charge and the size of the vesicles, are critical in regulating the effects of these vesicles on Aβ40 aggregation kinetics.

  17. Human FABP1 T94A variant enhances cholesterol uptake.

    PubMed

    Huang, Huan; McIntosh, Avery L; Landrock, Kerstin K; Landrock, Danilo; Storey, Stephen M; Martin, Gregory G; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2015-07-01

    Although expression of the human liver fatty acid binding protein (FABP1) T94A variant alters serum lipoprotein cholesterol levels in human subjects, nothing is known whereby the variant elicits these effects. This issue was addressed by in vitro cholesterol binding assays using purified recombinant wild-type (WT) FABP1 T94T and T94A variant proteins and in cultured primary human hepatocytes expressing the FABP1 T94T (genotyped as TT) or T94A (genotyped as CC) proteins. The human FABP1 T94A variant protein had 3-fold higher cholesterol-binding affinity than the WT FABP1 T94T as shown by NBD-cholesterol fluorescence binding assays and by cholesterol isothermal titration microcalorimetry (ITC) binding assays. CC variant hepatocytes also exhibited 30% higher total FABP1 protein. HDL- and LDL-mediated NBD-cholesterol uptake was faster in CC variant than TT WT human hepatocytes. VLDL-mediated uptake of NBD-cholesterol did not differ between CC and TT human hepatocytes. The increased HDL- and LDL-mediated NBD-cholesterol uptake was not associated with any significant change in mRNA levels of SCARB1, LDLR, CETP, and LCAT encoding the key proteins in lipoprotein cholesterol uptake. Thus, the increased HDL- and LDL-mediated NBD-cholesterol uptake by CC hepatocytes may be associated with higher affinity of T94A protein for cholesterol and/or increased total T94A protein level. PMID:25732850

  18. NADPH Oxidases in Heart Failure: Poachers or Gamekeepers?

    PubMed Central

    Zhang, Min; Perino, Alessia; Ghigo, Alessandra; Hirsch, Emilio

    2013-01-01

    Abstract Significance: Oxidative stress is involved in the pathogenesis of heart failure but clinical antioxidant trials have been unsuccessful. This may be because effects of reactive oxygen species (ROS) depend upon their source, location, and concentration. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) proteins generate ROS in a highly regulated fashion and modulate several components of the heart failure phenotype. Recent Advances: Two Nox isoforms, Nox2 and Nox4, are expressed in the heart. Studies using gene-modified mice deficient in Nox2 activity indicate that Nox2 activation contributes to angiotensin II–induced cardiomyocyte hypertrophy, atrial fibrillation, and the development of interstitial fibrosis but may also positively modulate physiological excitation-contraction coupling. Nox2 contributes to myocyte death under stress situations and plays important roles in postmyocardial infarction remodeling, in part by modulating matrix metalloprotease activity. In contrast to Nox2, Nox4 is constitutively active at a low level and induces protective effects in the heart under chronic stress, for example, by maintaining myocardial capillary density. However, high levels of Nox4 could have detrimental effects. Critical Issues: The effects of Nox proteins during the development of heart failure likely depend upon the isoform, activation level, and cellular distribution, and may include beneficial as well as detrimental effects. More needs to be learnt about the precise regulation of abundance and biochemical activity of these proteins in the heart as well as the downstream signaling pathways that they regulate. Future Directions: The development of specific approaches to target individual Nox isoforms and/or specific cell types may be important for the achievement of therapeutic efficacy in heart failure. Antioxid. Redox Signal. 18, 1024–1041. PMID:22747566

  19. High Cholesterol Deteriorates Bone Health: New Insights into Molecular Mechanisms

    PubMed Central

    Mandal, Chandi C.

    2015-01-01

    Many epidemiological studies show a positive connection between cardiovascular diseases and risk of osteoporosis, suggesting a role of hyperlipidemia and/or hypercholesterolemia in regulating osteoporosis. The majority of the studies indicated a correlation between high cholesterol and high LDL-cholesterol level with low bone mineral density, a strong predictor of osteoporosis. Similarly, bone metastasis is a serious complication of cancer for patients. Several epidemiological and basic studies have established that high cholesterol is associated with increased cancer risk. Moreover, osteoporotic bone environment predisposes the cancer cells for metastatic growth in the bone microenvironment. This review focuses on how cholesterol and cholesterol-lowering drugs (statins) regulate the functions of bone residential osteoblast and osteoclast cells to augment or to prevent bone deterioration. Moreover, this study provides an insight into molecular mechanisms of cholesterol-mediated bone deterioration. It also proposes a potential mechanism by which cellular cholesterol boosts cancer-induced bone metastasis. PMID:26557105

  20. The role of the lymphatic system in cholesterol transport

    PubMed Central

    Huang, Li-Hao; Elvington, Andrew; Randolph, Gwendalyn J.

    2015-01-01

    Reverse cholesterol transport (RCT) is the pathway for removal of peripheral tissue cholesterol and involves transport of cholesterol back to liver for excretion, starting from cellular cholesterol efflux facilitated by lipid-free apolipoprotein A1 (ApoA1) or other lipidated high-density lipoprotein (HDL) particles within the interstitial space. Extracellular cholesterol then is picked up and transported through the lymphatic vasculature before entering into bloodstream. There is increasing evidence supporting a role for enhanced macrophage cholesterol efflux and RCT in ameliorating atherosclerosis, and recent data suggest that these processes may serve as better diagnostic biomarkers than plasma HDL levels. Hence, it is important to better understand the processes governing ApoA1 and HDL influx into peripheral tissues from the bloodstream, modification and facilitation of cellular cholesterol removal within the interstitial space, and transport through the lymphatic vasculature. New findings will complement therapeutic strategies for the treatment of atherosclerotic vascular disease. PMID:26388772

  1. Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

    SciTech Connect

    Atlow, S.C.; Bonadonna-Aparo, L.; Klibanov, A.M.

    1984-01-01

    A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

  2. Step by Step: Eating To Lower Your High Blood Cholesterol. Revised.

    ERIC Educational Resources Information Center

    National Heart, Lung, and Blood Inst. (DHHS/NIH), Bethesda, MD.

    This booklet offers advice for adults who want to lower their blood cholesterol level. The first section, "What You Need To Know about High Blood Cholesterol," discusses blood cholesterol and why it matters, what cholesterol numbers mean, and what affects blood cholesterol levels. Section 2, "What You Need To Do To Lower Blood Cholesterol,"…

  3. Chewing Over Physiology Integration

    ERIC Educational Resources Information Center

    Abdulkader, Fernando; Azevedo-Martins, Anna Karenina; de Arcisio Miranda, Manoel; Brunaldi, Kellen

    2005-01-01

    An important challenge for both students and teachers of physiology is to integrate the differentareas in which physiological knowledge is didactically divided. In developing countries, such an issue is even more demanding, because budget restrictions often affect the physiology program with laboratory classes being the first on the list when it…

  4. Current status of NADPH oxidase research in cardiovascular pharmacology

    PubMed Central

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Castiñeiras-Landeira, María Isabel; Raposeiras-Roubín, Sergio; González-Juanatey, José R; Álvarez, Ezequiel

    2013-01-01

    The implications of reactive oxygen species in cardiovascular disease have been known for some decades. Rationally, therapeutic antioxidant strategies combating oxidative stress have been developed, but the results of clinical trials have not been as good as expected. Therefore, to move forward in the design of new therapeutic strategies for cardiovascular disease based on prevention of production of reactive oxygen species, steps must be taken on two fronts, ie, comprehension of reduction-oxidation signaling pathways and the pathophysiologic roles of reactive oxygen species, and development of new, less toxic, and more selective nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, to clarify both the role of each NADPH oxidase isoform and their utility in clinical practice. In this review, we analyze the value of NADPH oxidase as a therapeutic target for cardiovascular disease and the old and new pharmacologic agents or strategies to prevent NADPH oxidase activity. Some inhibitors and different direct or indirect approaches are available. Regarding direct NADPH oxidase inhibition, the specificity of NADPH oxidase is the focus of current investigations, whereas the chemical structure-activity relationship studies of known inhibitors have provided pharmacophore models with which to search for new molecules. From a general point of view, small-molecule inhibitors are preferred because of their hydrosolubility and oral bioavailability. However, other possibilities are not closed, with peptide inhibitors or monoclonal antibodies against NADPH oxidase isoforms continuing to be under investigation as well as the ongoing search for naturally occurring compounds. Likewise, some different approaches include inhibition of assembly of the NADPH oxidase complex, subcellular translocation, post-transductional modifications, calcium entry/release, electron transfer, and genetic expression. High-throughput screens for any of these activities could provide new

  5. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  6. CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

    PubMed

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

  7. CotA, a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

    PubMed Central

    Su, Jianmei; Bao, Peng; Bai, Tenglong; Deng, Lin; Wu, Hui; Liu, Fan; He, Jin

    2013-01-01

    Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10−6±0.21 M·min−1 and 0.32±0.02 s−1, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a

  8. Maternal exposure to di-(2-ethylhexyl) phthalate exposure deregulates blood pressure, adiposity, cholesterol metabolism and social interaction in mouse offspring.

    PubMed

    Lee, Kuan-I; Chiang, Chin-Wei; Lin, Hui-Ching; Zhao, Jin-Feng; Li, Cheng-Ta; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2016-05-01

    Long-term exposure to di-(2-ethylhexyl) phthalate (DEHP) is highly associated with carcinogenicity, fetotoxicity, psychological disorders and metabolic diseases, but the detrimental effects and mechanisms are not fully understood. We investigated the effect of exposing mouse mothers to DEHP, and the underlying mechanism, on blood pressure, obesity and cholesterol metabolism as well as psychological and learning behaviors in offspring. Tail-cuff plethysmography was used for blood pressure measurement; Western blot used was for phosphorylation and expression of protein; hematoxylin and eosin staining, Nissl staining and Golgi staining were used for histological examination. The serum levels of cholesterol, triglycerides and glucose were measured by blood biochemical analysis. Hepatic cholesterol and triglyceride levels were assessed by colorimetric assay kits. Offspring behaviors were evaluated by open-field activity, elevated plus maze, social preference test and Morris water maze. Maternal DEHP exposure deregulated the phosphorylation of endothelial nitric oxide synthase and upregulated angiotensin type 1 receptor in offspring, which led to increased blood pressure. It led to obesity in offspring by increasing the size of adipocytes in white adipose tissue and number of adipocytes in brown adipose tissue. It increased the serum level of cholesterol in offspring by decreasing the hepatic capacity for cholesterol clearance. The impaired social interaction ability induced by maternal DEHP exposure might be due to abnormal neuronal development. Collectively, our findings provide new evidence that maternal exposure to DEHP has a lasting effect on the physiological functions of the vascular system, adipose tissue and nerve system in offspring.

  9. Multilayered Polyelectrolyte Microcapsules: Interaction with the Enzyme Cytochrome C Oxidase

    PubMed Central

    Pastorino, Laura; Dellacasa, Elena; Noor, Mohamed R.; Soulimane, Tewfik; Bianchini, Paolo; D'Autilia, Francesca; Antipov, Alexei; Diaspro, Alberto; Tofail, Syed A. M.; Ruggiero, Carmelina

    2014-01-01

    Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties. PMID:25372607

  10. NADPH oxidase deficiency in X-linked chronic granulomatous disease.

    PubMed Central

    Hohn, D C; Lehrer, R I

    1975-01-01

    We measured the cyanide-insensitive pyridine nucleotide oxidase activity of fractionated resting and phagocytic neutrophils from 11 normal donors, 1 patient with hereditary deficiency of myeloperoxidase, and 7 patients with X-linked chronic granulomatous disease (CGD). When measured under optimal conditions (at pH 5.5 and in the presence of 0.5 mM Mn++), NADPH oxidase activity increased fourfold with phagocytosis and was six-fold higher than with NADH. Phagocytic neutrophils from patients with CGD were markedly deficient in NADPH oxidase activity. Images PMID:235560

  11. Identification of yeasts from clinical specimens by oxidase test.

    PubMed

    Kumar, S; Arora, B S; Mathur, M D

    2000-10-01

    A total of 100 yeasts and yeast like fungi isolates from clinical specimens were negative for oxidase production on Sabouraud dextrose agar. When grown on Columbia agar, chocolate agar, tryptose agar, Mueller-Hinton agar, brain heart infusion and a medium resembling Sabouraud's dextrose agar but with starch instead of dextrose, all the isolate of Candida albicans (55), C. guilliermondii (6), C. parapsilosis (14), C. tropicalis (6), C. pseudotropicalis (6) and Crytococcus neoformans (2) were positive for oxidase producation. Torulopsis glabrata (2), Saccharomyces cervisiae (2) and two out of seven isolates of C. krusei were negative for oxidase test. PMID:11344606

  12. Functional analysis of polyphenol oxidases by antisense/sense technology.

    PubMed

    Thipyapong, Piyada; Stout, Michael J; Attajarusit, Jutharat

    2007-01-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting

  13. Increased hepatic cholesterol esterification with essential fatty acid deficiency (EFAD): relationship to plasma lipoprotein (LP) cholesterol content

    SciTech Connect

    Ney, D.M.; Ziboh, V.A.; Schneeman, B.O.

    1986-03-01

    EFAD in the rat is associated with hepatic accumulation of esterified cholesterol and altered distribution of cholesterol between plasma and hepatic tissue. Little is known regarding the impact of EFAD on LP composition. To determine the relationship between hepatic cholesterol esterification and plasma lP composition in control (C) and EFAD male Wistar rats, the authors induced EFAD with continuous intragastric (IG) infusion of EFA-free solutions containing 3.5% of calories as triolein for 7 and 14 days. C animals received IG infusion of solutions containing 3.5% of calories as linoleic acid. Data in the EFAD groups reveal: (i) marked decreases in hepatic EFAs and increases in monoenoic acids; (ii) progressive increases in hepatic content of triglyceride and esterified cholesterol with 7 and 14 days of feeding; (iii) assay of acyl CoA:cholesterol acyltransferase activity in hepatic tissue using /sup 14/C-cholesterol demonstrates an increase in hepatic cholesterol esterification when compared to C animals. Increased hepatic cholesterol esterification correlates with elevated levels of esterified cholesterol in plasma VLDL and HDL particles. These data indicate that the elevated levels of cholesterol esters in LP particles is due, at least in part, to increased hepatic cholesterol esterification with EFAD.

  14. Alternative Oxidase Expression in the Mouse Enables Bypassing Cytochrome c Oxidase Blockade and Limits Mitochondrial ROS Overproduction

    PubMed Central

    El-Khoury, Riyad; Dufour, Eric; Rak, Malgorzata; Ramanantsoa, Nelina; Grandchamp, Nicolas; Csaba, Zsolt; Duvillié, Bertrand; Bénit, Paule; Gallego, Jorge; Gressens, Pierre; Sarkis, Chamsy; Jacobs, Howard T.; Rustin, Pierre

    2013-01-01

    Cyanide-resistant non-phosphorylating respiration is known in mitochondria from plants, fungi, and microorganisms but is absent in mammals. It results from the activity of an alternative oxidase (AOX) that conveys electrons directly from the respiratory chain (RC) ubiquinol pool to oxygen. AOX thus provides a bypath that releases constraints on the cytochrome pathway and prevents the over-reduction of the ubiquinone pool, a major source of superoxide. RC dysfunctions and deleterious superoxide overproduction are recurrent themes in human pathologies, ranging from neurodegenerative diseases to cancer, and may be instrumental in ageing. Thus, preventing RC blockade and excess superoxide production by means of AOX should be of considerable interest. However, because of its energy-dissipating properties, AOX might produce deleterious effects of its own in mammals. Here we show that AOX can be safely expressed in the mouse (MitAOX), with major physiological parameters being unaffected. It neither disrupted the activity of other RC components nor decreased oxidative phosphorylation in isolated mitochondria. It conferred cyanide-resistance to mitochondrial substrate oxidation and decreased reactive oxygen species (ROS) production upon RC blockade. Accordingly, AOX expression was able to support cyanide-resistant respiration by intact organs and to afford prolonged protection against a lethal concentration of gaseous cyanide in whole animals. Taken together, these results indicate that AOX expression in the mouse is innocuous and permits to overcome a RC blockade, while reducing associated oxidative insult. Therefore, the MitAOX mice represent a valuable tool in order to investigate the ability of AOX to counteract the panoply of mitochondrial-inherited diseases originating from oxidative phosphorylation defects. PMID:23300486

  15. Aldehyde Oxidase Functions as a Superoxide Generating NADH Oxidase: An Important Redox Regulated Pathway of Cellular Oxygen Radical Formation

    PubMed Central

    Kundu, Tapan K.; Velayutham, Murugesan; Zweier, Jay L.

    2012-01-01

    The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress. Utilizing electron paramagnetic resonance spin trapping we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH, and the effects of inhibitors of AO on NADH-mediated superoxide ( O2•−) generation. NADH was found to be a good substrate for AO with apparent Km and Vmax values of 29μM and 12 nmol min−1 mg−1, respectively. From O2•− generation measurements by cytochrome c reduction the apparent Km and Vmax values of NADH for AO were 11 μM and 15 nmol min−1 mg−1, respectively. With NADH oxidation by AO, ≥65% of the total electron flux led to O2•− generation. Diphenyleneiodonium completely inhibited AO-mediated O2•− production confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O2•− generation were studied with amidone the most potent exerting complete inhibition at 100 μM concentration, while 150 μM menadione, raloxifene or β-estradiol led to 81%, 46% or 26% inhibition, respectively. From the kinetic data, the levels of AO and NADH, O2•− production was estimated to be ~89 and ~4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions. Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis. PMID:22404107

  16. Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis*

    PubMed Central

    Capyk, Jenna K.; Casabon, Israël; Gruninger, Robert; Strynadka, Natalie C.; Eltis, Lindsay D.

    2011-01-01

    Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (kcat/Km) of KshAB for the CoA thioester substrates was 20–30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent KmO2 was 90 ± 10 μm in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ1 ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism. PMID:21987574

  17. A New Model of Reverse Cholesterol Transport: EnTICEing Strategies to Stimulate Intestinal Cholesterol Excretion

    PubMed Central

    Temel, Ryan E.; Brown, J. Mark

    2015-01-01

    Cardiovascular disease (CVD) remains the largest cause of mortality in most developed countries. Although recent failed clinical trials and Mendelian randomization studies have called into question the high density lipoprotein (HDL) hypothesis, it remains well accepted that stimulating the process of reverse cholesterol transport (RCT) can prevent or even regress atherosclerosis. The prevailing model for RCT is that cholesterol from the artery wall must be delivered to the liver where it is secreted into bile before leaving the body through fecal excretion. However, many studies have demonstrated that RCT can proceed through a non-biliary pathway known as transintestinal cholesterol excretion (TICE). The goal of this review is to discuss the current state of knowledge of the TICE pathway, with emphasis on points of therapeutic intervention. PMID:25930707

  18. [Evolution of evolutionary physiology].

    PubMed

    Natochin, Iu V

    2008-09-01

    In 19th century and at the beginning 20th century, reports appeared in the field of comparative and ontogenetic physiology and the value of these methods for understanding of evolution of functions. The term "evolutionary physiology" was suggested by A. N. Severtsov in 1914. In the beginning of 30s, in the USSR, laboratories for researches in problems of evolutionary physiology were created, the results of these researches having been published. In 1956 in Leningrad, the Institute of Evolutionary Physiology was founded by L. A. Orbeli. He formulates the goals and methods of evolutionary physiology. In the following half a century, the evolutionary physiology was actively developed. The evolutionary physiology solves problems of evolution of function of functions evolution, often involving methods of adjacent sciences, including biochemistry, morphology, molecular biology.

  19. Direct Regulation of Prokaryotic Kir Channel by Cholesterol*

    PubMed Central

    Singh, Dev K.; Rosenhouse-Dantsker, Avia; Nichols, Colin G.; Enkvetchakul, Decha; Levitan, Irena

    2009-01-01

    Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by 86Rb+ uptake. Our results show that 86Rb+ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses 86Rb+ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated 86Rb+ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific. PMID:19740741

  20. Control of Angiogenesis by AIBP-mediated Cholesterol Efflux

    PubMed Central

    Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vázquez, Jesús; Li, Andrew C.; Miller, Yury I.

    2013-01-01

    Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis. PMID:23719382