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Sample records for chloroplastic carbonic anhydrase

  1. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    In a new assay of carbonic anhydrase, NaH14CO3 solution at the bottom of a sealed vessel releases 14CO3 which diffuses to the top of the vessel to be assimilated by actively photosynthesizing Chlamydomonas cells. The assay is initiated by illuminating cells and stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid stable radioactivity above the uncatalyzed background level. With bovine carbonic anhydrase, 1.5 Wilbur Anderson Unit (WAU) can be consistantly measured at 5-6 fold above background. Sonicated whole cells of air adapted wild type (+)gave 741.1 ± 12.4 WAU/mg chl. Intact washed cells of mixotrophically grown wall-less mutant CWD(-) and a high CO2 requiring wall-less double mutant CIA-3/CW15 (-) gave 7.1 ± 1.9 and 2.8 ± 7.8 WAU/mg chl respectively. Chloroplasts isolated from CWD and CIA-3/CW15 and subsequently disrupted gave 64.0 ± 14.7 and 2.8 ± 3.2 WAU/mg chl respectively. Chloroplast sonicate from another wall-less mutant CW15(-) gave activity comparable to CWD. Thus on a chlorophyll basis, enzyme activity in chloroplasts from mixotrophically grown cells is about 1/10th of the level found in air adapted wild type cells. CIA-3 seems to lack this activity

  2. Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Stefan Burén

    Full Text Available BACKGROUND: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. CONCLUSIONS/SIGNIFICANCE: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native

  3. Natural products that inhibit carbonic anhydrase.

    Science.gov (United States)

    Poulsen, Sally-Ann; Davis, Rohan A

    2014-01-01

    The chemical diversity, binding specificity and propensity to interact with biological targets has inspired many researchers to utilize natural products as molecular probes. Almost all reported carbonic anhydrase inhibitors comprise a zinc binding group in their structure of which the primary sulfonamide moiety (-SO2NH2) is the foremost example and to a lesser extent the primary sulfamate (-O-SO2NH2) and sulfamide (-NH-SO2NH2) groups. Natural products that comprise these zinc binding groups in their structure are however rare and relatively few natural products have been explored as a source for novel carbonic anhydrase inhibitors. This chapter will highlight the recent and growing interest in carbonic anhydrase inhibitors sourced from nature, demonstrating that natural product chemical space presents a rich source of potential alternate chemotypes for the discovery of novel drug-like carbonic anhydrase inhibitors. PMID:24146386

  4. Accelerating Mineral Carbonation Using Carbonic Anhydrase.

    Science.gov (United States)

    Power, Ian M; Harrison, Anna L; Dipple, Gregory M

    2016-03-01

    Carbonic anhydrase (CA) enzymes have gained considerable attention for their potential use in carbon dioxide (CO2) capture technologies because they are able to catalyze rapidly the interconversion of aqueous CO2 and bicarbonate. However, there are challenges for widespread implementation including the need to develop mineralization process routes for permanent carbon storage. Mineral carbonation of highly reactive feedstocks may be limited by the supply rate of CO2. This rate limitation can be directly addressed by incorporating enzyme-catalyzed CO2 hydration. This study examined the effects of bovine carbonic anhydrase (BCA) and CO2-rich gas streams on the carbonation rate of brucite [Mg(OH)2], a highly reactive mineral. Alkaline brucite slurries were amended with BCA and supplied with 10% CO2 gas while aqueous chemistry and solids were monitored throughout the experiments (hours to days). In comparison to controls, brucite carbonation using BCA was accelerated by up to 240%. Nesquehonite [MgCO3·3H2O] precipitation limited the accumulation of hydrated CO2 species, apparently preventing BCA from catalyzing the dehydration reaction. Geochemical models reproduce observed reaction progress in all experiments, revealing a linear correlation between CO2 uptake and carbonation rate. Data demonstrates that carbonation in BCA-amended reactors remained limited by CO2 supply, implying further acceleration is possible. PMID:26829491

  5. Carbonic anhydrase inhibitors drug design.

    Science.gov (United States)

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported. PMID:24146385

  6. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  7. Heterocyclic compounds as carbonic anhydrase inhibitor.

    Science.gov (United States)

    Husain, Asif; Madhesia, Diwakar

    2012-12-01

    The carbonic anhydrases (CAs, EC 4.2.1.1) constitute interesting targets for the design of pharmacological agents useful in the treatment or prevention of a variety of disorders such as, glaucoma, acid-base disequilibria, epilepsy, and other neuromuscular diseases, altitude sickness, edema, and obesity. A quite new and unexpected application of the CA inhibitors (CAIs) is with regard to their potential use in the management (imaging and treatment) of hypoxic tumors. A series of sulfonamides, including some clinically used derivatives like acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug, as well as the sulfamate antiepileptic drug topiramate have been reported to inhibit various human carbonic anhydrase isozyme. Various heterocyclic sulfonamides have been reported in this review with their potency to inhibit different carbonic anhydrases isozymes. PMID:21981003

  8. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

    Science.gov (United States)

    Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T.; Tambutté, Sylvie

    2016-01-01

    Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity. PMID:27271641

  9. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    Science.gov (United States)

    Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T; Tambutté, Sylvie

    2016-01-01

    Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

  10. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

    Directory of Open Access Journals (Sweden)

    Didier Zoccola

    2016-06-01

    Full Text Available Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1 a change in gene expression under OA (2 an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.

  11. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    Science.gov (United States)

    Zoccola, Didier; Innocenti, Alessio; Bertucci, Anthony; Tambutté, Eric; Supuran, Claudiu T; Tambutté, Sylvie

    2016-01-01

    Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity. PMID:27271641

  12. The Cellular Physiology of Carbonic Anhydrases

    Directory of Open Access Journals (Sweden)

    Breton S

    2001-07-01

    Full Text Available Carbonic anhydrases are zinc metalloenzymes that catalyze the reversible hydration of CO(2 to form HCO(3(- and protons according to the following reaction: CO(2 + H(2O = H(2CO(3 = HCO(3(- + H(+. The first reaction is catalyzed by carbonic anhydrase and the second reaction occurs instantaneously. The carbonic anhydrase (CA gene family includes ten enzymatically active members, which are major players in many physiological processes, including renal and male reproductive tract acidification, bone resorption, respiration, gluconeogenesis, signal transduction, and formation of gastric acid. The newly identified CA IX (previously called MN and CA XII are related to cell proliferation and oncogenesis. Carbonic anhydrase isozymes have different kinetic properties and they are present in various tissues and in various cell compartments. CA I, II, III and VII are cytoplasmic, CA V is mitochondrial, and CA VI is present in salivary secretions. CA IV, IX, XII and XIV are membrane proteins: CA IV is a glycosyl-phosphatidylinositol-anchored protein, and CA IX, XII and XIV are transmembrane proteins. The present work will focus on the roles of CA II and CA IV in transepithelial proton secretion and bicarbonate reabsorption processes. The localization of these isoforms in selected epithelia that are involved in net acid/base transport, such as kidney proximal tubules and collecting ducts, and tubules from the male reproductive tract will be reviewed.

  13. Evolution of carbonic anhydrase in C4 plants.

    Science.gov (United States)

    Ludwig, Martha

    2016-06-01

    During the evolution of C4 photosynthesis, the intracellular location with most carbonic anhydrase (CA) activity has changed. In Flaveria, the loss of the sequence encoding a chloroplast transit peptide from an ancestral C3 CA ortholog confined the C4 isoform to the mesophyll cell cytosol. Recent studies indicate that sequence elements and histone modifications controlling the expression of C4-associated CAs were likely present in the C3 ancestral chromatin, enabling the evolution of the C4 pathway. Almost complete abolishment of maize CA activity yields no obvious phenotype at ambient CO2 levels. This contrasts with results for Flaveria CA mutants, and has opened discussion on the role of CA in the C4 carbon concentrating mechanism.

  14. Hyperkalaemia induced by carbonic anhydrase inhibitor.

    OpenAIRE

    Wakabayashi, Y.

    1991-01-01

    An 81-year-old man developed hyperkalaemic and hyperchloraemic metabolic acidosis following treatment with a carbonic anhydrase inhibitor for his glaucoma. He had mild renal failure and selective aldosterone deficiency was confirmed. In this case the treatment did not lead to hypokalaemia because of the limited potassium secretory capacity in the renal tubules from selective aldosterone deficiency; rather, it may have led to hyperkalaemia because metabolic acidosis induced by the carbonic anh...

  15. Thermostable Carbonic Anhydrases in Biotechnological Applications

    OpenAIRE

    Anna Di Fiore; Vincenzo Alterio; Simona M. Monti; Giuseppina De Simone; Katia D'Ambrosio

    2015-01-01

    Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques,...

  16. Inhibitory Effect of Furosemide on Carbonic Anhydrase

    Institute of Scientific and Technical Information of China (English)

    CUI Jianli; ZHAO Tongjin; JIANG Yan; ZHOU Haimeng

    2006-01-01

    This study investigated the inhibitory effect of a high efficiency diuretic, furosemide, on carbonic anhydrase (CA). First, comparing the inhibitory effect of acetazolamide, a low efficiency diuretic, on CA, shows that furosemide or acetazolamide can quickly make CA inactive when its concentration is close to the enzyme concentration, different from the usual inhibitory kinetics in which the concentration of the inhibitor is far higher than the enzyme concentration. Secondly, the reaction of the enzyme indicates that the inhibitory effect of furosemide or acetazolamide on carbonic anhydrase is quickly reversible. Finally, the degree of the inhibitory effect of furosemide and of acetazolamide on CA are compared. The results show that furosemide inhibits CA less than acetazolamide.

  17. Non-Classical Inhibition of Carbonic Anhydrase

    Science.gov (United States)

    Lomelino, Carrie L.; Supuran, Claudiu T.; McKenna, Robert

    2016-01-01

    Specific isoforms from the carbonic anhydrase (CA) family of zinc metalloenzymes have been associated with a variety of diseases. Isoform-specific carbonic anhydrase inhibitors (CAIs) are therefore a major focus of attention for specific disease treatments. Classical CAIs, primarily sulfonamide-based compounds and their bioisosteres, are examined as antiglaucoma, antiepileptic, antiobesity, antineuropathic pain and anticancer compounds. However, many sulfonamide compounds inhibit all CA isoforms nonspecifically, diluting drug effectiveness and causing undesired side effects due to off-target inhibition. In addition, a small but significant percentage of the general population cannot be treated with sulfonamide-based compounds due to a sulfa allergy. Therefore, CAIs must be developed that are not only isoform specific, but also non-classical, i.e. not based on sulfonamides, sulfamates, or sulfamides. This review covers the classes of non-classical CAIs and the recent advances in the development of isoform-specific inhibitors based on phenols, polyamines, coumarins and their derivatives. PMID:27438828

  18. Carbonic Anhydrases and Their Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Robert McKenna

    2013-08-01

    Full Text Available The carbonic anhydrases (CAs are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II has a catalytic efficiency of 108 M−1 s−1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development.

  19. Carbonic Anhydrase and Metalloderivatives: A Bioinorganic Chemistry Study

    Science.gov (United States)

    McQuate, Robert S.

    1977-01-01

    Discusses selected bioinorganic aspects of carbonic anhydrase and describes experiments that will reinforce the students' understanding of the presence and essential role that metal ions have in some biological systems. (SL)

  20. Future Perspective in Carbonic Anhydrase Inhibitors and its Drugs

    OpenAIRE

    S.Petchimuthu; Dr. N. Narayanan

    2013-01-01

    Through this review it is contemplated that carbonic anhydrase inhibitors, were a traditional drugs of choice for the treatment of glaucoma with a myriad of side effects and inadequate topical effectiveness, may be formulated into a topically effective agent by utilizing various newer formulation approaches of ocular drug delivery. Even though the carbonic anhydrase inhibitor, acetazolamide (ACZ) has a poor solubility and penetration power (BCS Class IV), various studies mentioned in the revi...

  1. Structure and function of carbonic anhydrases.

    Science.gov (United States)

    Supuran, Claudiu T

    2016-07-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyse the interconversion between CO2 and bicarbonate as well as other hydrolytic reactions. Among the six genetic families known to date, the α-, β-, γ-, δ-, ζ- and η-CAs, detailed kinetic and X-ray crystallographic studies have allowed a deep understanding of the structure-function relationship in this superfamily of proteins. A metal hydroxide nucleophilic species of the enzyme, and a unique active site architecture, with half of it hydrophilic and the opposing part hydrophobic, allow these enzymes to act as some of the most effective catalysts known in Nature. The CA activation and inhibition mechanisms are also known in detail, with a large number of new inhibitor classes being described in the last years. Apart from the zinc binders, some classes of inhibitors anchor to the metal ion coordinated nucleophile, others occlude the entrance of the active site cavity and more recently, compounds binding outside the active site were described. CA inhibition has therapeutic applications for drugs acting as diuretics, antiepileptics, antiglaucoma, antiobesity and antitumour agents. Targeting such enzymes from pathogens may lead to novel anti-infectives. Successful structure-based drug design campaigns allowed the discovery of highly isoform selective CA inhibitors (CAIs), which may lead to a new generation of drugs targeting these widespread enzymes. The use of CAs in CO2 capture processes for mitigating the global temperature rise has also been investigated more recently. PMID:27407171

  2. How many carbonic anhydrase inhibition mechanisms exist?

    Science.gov (United States)

    Supuran, Claudiu T

    2016-01-01

    Six genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) were described to date. Inhibition of CAs has pharmacologic applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents. New classes of CA inhibitors (CAIs) were described in the last decade with enzyme inhibition mechanisms differing considerably from the classical inhibitors of the sulfonamide or anion type. Five different CA inhibition mechanisms are known: (i) the zinc binders coordinate to the catalytically crucial Zn(II) ion from the enzyme active site, with the metal in tetrahedral or trigonal bipyramidal geometries. Sulfonamides and their isosters, most anions, dithiocarbamates and their isosters, carboxylates, and hydroxamates bind in this way; (ii) inhibitors that anchor to the zinc-coordinated water molecule/hydroxide ion (phenols, carboxylates, polyamines, 2-thioxocoumarins, sulfocoumarins); (iii) inhibitors which occlude the entrance to the active site cavity (coumarins and their isosters), this binding site coinciding with that where CA activators bind; (iv) compounds which bind out of the active site cavity (a carboxylic acid derivative was seen to inhibit CA in this manner), and (v) compounds for which the inhibition mechanism is not known, among which the secondary/tertiary sulfonamides as well as imatinib/nilotinib are the most investigated examples. As CAIs are used clinically in many pathologies, with a sulfonamide inhibitor (SLC-0111) in Phase I clinical trials for the management of metastatic solid tumors, this review updates the recent findings in the field which may be useful for a structure-based drug design approach of more selective/potent modulators of the activity of these enzymes. PMID:26619898

  3. Carbonic anhydrases as targets for medicinal chemistry.

    Science.gov (United States)

    Supuran, Claudiu T; Scozzafava, Andrea

    2007-07-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc enzymes acting as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate. 16 different alpha-CA isoforms were isolated in mammals, where they play crucial physiological roles. Some of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is secreted in saliva and milk. Three acatalytic forms are also known, the CA related proteins (CARP), CARP VIII, CARP X and CARP XI. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited primarily by two classes of compounds: the metal complexing anions and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO(2)NH(2) (R=aryl; hetaryl; perhaloalkyl; X=nothing, O or NH). Several important physiological and physio-pathological functions are played by CAs present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms represents an attractive goal for the design of inhibitors with biomedical applications. Indeed, CA inhibitors are clinically used as

  4. Enzymes for carbon sequestration: neutron crystallographic studies of carbonic anhydrase

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, S. Z., E-mail: zfisher@lanl.gov; Kovalevsky, A. Y. [Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States); Domsic, J. [Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States); Mustyakimov, M. [Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States); Silverman, D. N. [Department of Pharmacology and Therapeutics, PO Box 100267, University of Florida, Gainesville, FL 32610 (United States); McKenna, R. [Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States); Langan, P. [Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States)

    2010-11-01

    The first neutron crystal structure of carbonic anhydrase is presented. The structure reveals interesting and unexpected features of the active site that affect catalysis. Carbonic anhydrase (CA) is a ubiquitous metalloenzyme that catalyzes the reversible hydration of CO{sub 2} to form HCO{sub 3}{sup −} and H{sup +} using a Zn–hydroxide mechanism. The first part of catalysis involves CO{sub 2} hydration, while the second part deals with removing the excess proton that is formed during the first step. Proton transfer (PT) is thought to occur through a well ordered hydrogen-bonded network of waters that stretches from the metal center of CA to an internal proton shuttle, His64. These waters are oriented and ordered through a series of hydrogen-bonding interactions to hydrophilic residues that line the active site of CA. Neutron studies were conducted on wild-type human CA isoform II (HCA II) in order to better understand the nature and the orientation of the Zn-bound solvent (ZS), the charged state and conformation of His64, the hydrogen-bonding patterns and orientations of the water molecules that mediate PT and the ionization of hydrophilic residues in the active site that interact with the water network. Several interesting and unexpected features in the active site were observed which have implications for how PT proceeds in CA.

  5. Carbonic Anhydrase II Deficiency in a Saudi Woman

    OpenAIRE

    Alhuzaim, Omar N; Almohareb, Ohoud M; Safiya M. Sherbeeni

    2015-01-01

    OBJECTIVE Carbonic anhydrase (CA) II deficiency is a rare autosomal recessive disorder caused by mutation in the CA II gene that leads to osteopetrosis, renal tubular acidosis (RTA), and cerebral calcification. Our aim is to present a patient with the classic triad of CA II deficiency syndrome to enhance the awareness about this rare syndrome. METHODS We describe the clinical and radiological findings of a Saudi woman patient with CA II deficiency syndrome. RESULTS A Saudi woman in her 20s pr...

  6. Structural analysis of inhibitor binding to human carbonic anhydrase II.

    OpenAIRE

    Boriack-Sjodin, P. A.; Zeitlin, S; Chen, H H; Crenshaw, L.; Gross, S.; Dantanarayana, A.; P. Delgado; May, J. A.; Dean, T.; Christianson, D. W.

    1998-01-01

    X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bi...

  7. Electropolymerized carbonic anhydrase immobilization for carbon dioxide capture.

    Science.gov (United States)

    Merle, Geraldine; Fradette, Sylvie; Madore, Eric; Barralet, Jake E

    2014-06-17

    Biomimetic carbonation carried out with carbonic anhydrase (CA) in CO2-absorbing solutions, such as methyldiethanolamine (MDEA), is one approach that has been developed to accelerate the capture of CO2. However, there are several practical issues, such as high cost and limited enzyme stability, that need to be overcome. In this study, the capacity of CA immobilization on a porous solid support was studied to improve the instability in the tertiary amine solvent. We have shown that a 63% porosity macroporous carbon foam support makes separation and reuse facile and allows for an efficient supply and presentation of CO2 to an aqueous solvent and the enzyme catalytic center. These enzymatic supports conserved 40% of their initial activity after 42 days at 70 °C in an amine solvent, whereas the free enzyme shows no activity after 1 h in the same conditions. In this work, we have overcome the technical barrier associated with the recovery of the biocatalyst after operation, and most of all, these electropolymerized enzymatic supports have shown a remarkable increase of thermal stability in an amine-based CO2 sequestration solvent. PMID:24856780

  8. Future Perspective in Carbonic Anhydrase Inhibitors and its Drugs

    Directory of Open Access Journals (Sweden)

    S.Petchimuthu

    2013-09-01

    Full Text Available Through this review it is contemplated that carbonic anhydrase inhibitors, were a traditional drugs of choice for the treatment of glaucoma with a myriad of side effects and inadequate topical effectiveness, may be formulated into a topically effective agent by utilizing various newer formulation approaches of ocular drug delivery. Even though the carbonic anhydrase inhibitor, acetazolamide (ACZ has a poor solubility and penetration power (BCS Class IV, various studies mentioned in the review indicate that it is possible to successfully formulate topically effective ACZ by using:(i High concentration of the drug, (ii Surfactant gel preparations of ACZ, (iii ACZ loaded into liposomes, (iv Cyclodextrins to increase the solubility and hence bioavailability of ACZ, and Viscolyzers and other polymers either alone or in combination with cyclodextrins. With the advent of newer topical carbonic anhydrase inhibitors (CAIs like dorzolamide and brinzolamide, a localized effect with fewer side effects is expected.But whenever absorbed systemically, a similar range of adverse effects (attributable to sulphonamides may occur upon use. Furthermore, oral ACZ is reported to be more physiologically effective than 2% dorzolamide hydrochloridead ministered topically, even though in isolated tissues dorzolamide appears to be the most active as it shows the lowest IC50 values for CA-II and CA-IV. Hence, there exists considerable scope for the development of more/equally effective and inexpensive topically effective formulations of ACZ. The use of various formulation technologies discussed in this review can provide a fresh impetus to research in this area.

  9. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    OpenAIRE

    Supuran, Claudiu T.

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3) × 105 s−1 and kcat/KM ...

  10. Density functional theory study of proton transfer in carbonic anhydrase

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lidong; XIE Daiqian

    2005-01-01

    Proton transfer in carbonic anhydrase II has been studied at the B3LYP/6-31G(D) level. The active site model consists of the zinc ion, four histidine residues, two threonine residues, and three water molecules. Our calculations showed that the proton of the zinc-bound water molecule could be transferred to the nearest water molecule and an intermediate containing H3O+ is then formed. The intermediate is only 1.3 kJ·mol-1 above the reactant complex, whereas the barrier height for the proton transfer is about 8.1 kJ·mol-1.

  11. Sarcoidosis patient: an unexpected reaction to carbonic anhydrase enzyme inhibitor

    OpenAIRE

    Khedr, Yahya A H; Khedr, Abdulla H

    2013-01-01

    Ocular diseases are very common in many of the systemic diseases such as sarcoidosis, and may sometimes be the presenting symptom of the disease. In this case report, we present an unusual reaction of the sarcoid granuloma to carbonic anhydrase enzyme inhibitors (CAIs), which was encountered in a patient with ocular sarcoidosis. This observation was taken after a 2-week interval between a CT scan orbits and an MRI orbits which showed a decrease in size from 4×3×4 cm to 2.5×2.5×2 cm, respectiv...

  12. The effects of some bromophenols on human carbonic anhydrase isoenzymes.

    Science.gov (United States)

    Taslimi, Parham; Gülçin, İlhami; Öztaşkın, Necla; Çetinkaya, Yasin; Göksu, Süleyman; Alwasel, Saleh H; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrases (CAs, EC 4.2.1.1), which are involved in a variety of physiological and pathological processes, are ubiquitous metalloenzymes mainly catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate ([Formula: see text]) and proton (H(+)). In this study, a dozen of bromophenol derivatives (1-12) were evaluated as metalloenzyme CA (EC 4.2.1.1) inhibitors against the human carbonic anhydrase isoenzymes I and II (hCA I and II). Cytosolic hCA I and II isoenzymes were effectively inhibited by bromophenol derivatives (1-12) with Kis in the low nanomolar range of 1.85 ± 0.58 to 5.04 ± 1.46 nM against hCA I and in the range of 2.01 ± 0.52 to 2.94 ± 1.31 nM against hCA II, respectively. PMID:26133541

  13. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors

    DEFF Research Database (Denmark)

    Stefánsson, E; Jensen, P K; Eysteinsson, T;

    1999-01-01

    To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture....

  14. Carbonic Anhydrase: An Efficient Enzyme with Possible Global Implications

    Directory of Open Access Journals (Sweden)

    Christopher D. Boone

    2013-01-01

    Full Text Available As the global atmospheric emissions of carbon dioxide (CO2 and other greenhouse gases continue to grow to record-setting levels, so do the demands for an efficient and inexpensive carbon sequestration system. Concurrently, the first-world dependence on crude oil and natural gas provokes concerns for long-term availability and emphasizes the need for alternative fuel sources. At the forefront of both of these research areas are a family of enzymes known as the carbonic anhydrases (CAs, which reversibly catalyze the hydration of CO2 into bicarbonate. CAs are among the fastest enzymes known, which have a maximum catalytic efficiency approaching the diffusion limit of 108 M−1s−1. As such, CAs are being utilized in various industrial and research settings to help lower CO2 atmospheric emissions and promote biofuel production. This review will highlight some of the recent accomplishments in these areas along with a discussion on their current limitations.

  15. Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ruben Postel

    Full Text Available The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5 mutation, collapse of fins (cof, which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.

  16. Optic nerve oxygen tension in pigs and the effect of carbonic anhydrase inhibitors

    DEFF Research Database (Denmark)

    Stefánsson, E; Jensen, P K; Eysteinsson, T;

    1999-01-01

    To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture.......To evaluate how the oxygen tension of the optic nerve (ONP(O)2) is affected by the administration of the carbonic anhydrase inhibitors dorzolamide and acetazolamide and by alterations in oxygen and carbon dioxide in the breathing mixture....

  17. Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

    Science.gov (United States)

    Guilloton, M B; Korte, J J; Lamblin, A F; Fuchs, J A; Anderson, P M

    1992-02-25

    The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.

  18. Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours

    Institute of Scientific and Technical Information of China (English)

    Antti J. Kivel(a); Jyrki Kivel(a); Juha Saarnio; Seppo Parkkila

    2005-01-01

    Carbonic anhydrases (CAs) catalyse the hydration of CO2to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. This review is focused on the distribution and role of CA isoenzymes in both normal and pathological gastrointestinal (GI) tract tissues. It has been known for many years that CAs are widely present in the GI tract and play important roles in several physiological functions such as production of saliva, gastric acid, bile, and pancreatic juice as well as in absorption of salt and water in intestine. New information suggests that these enzymes participate in several processes that were not envisioned earlier. Especially, the recent reports on plasma membranebound isoenzymes Ⅸ and Ⅻ have raised considerable interest since they were reported to participate in cancer invasion and spread. They are induced by tumour hypoxia and may also play a role in von Hippel-Lindau (VHL)-mediated carcinogenesis.

  19. Bortezomib inhibits bacterial and fungal β-carbonic anhydrases.

    Science.gov (United States)

    Supuran, Claudiu T

    2016-09-15

    Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from pathogenic fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, Malassezia globosa) and bacteria (three isoforms from Mycobacterium tuberculosis, Rv3273, Rv1284 and Rv3588), as well from the insect Drosophila melanogaster (DmeCA) and the plant Flaveria bidentis (FbiCA1) with the boronic acid peptidomimetic proteosome inhibitor bortezomib was investigated. Bortezomib was a micromolar inhibitor of all these enzymes, with KIs ranging between 1.12 and 11.30μM. Based on recent crystallographic data it is hypothesized that the B(OH)2 moiety of the inhibitor is directly coordinated to the zinc ion from the enzyme active site. The class of boronic acids, an under-investigated type of CA inhibitors, may lead to the development of anti-infectives with a novel mechanism of action, based on the pathogenic organisms CA inhibition. PMID:27469982

  20. Carbonic Anhydrase: In the Driver's Seat for Bicarbonate Transport

    Directory of Open Access Journals (Sweden)

    Sterling D

    2001-07-01

    Full Text Available Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO(2 + H(2O = HCO(3(- + H(+. These enzymes therefore both produce HCO(3(- for transport across membranes and consume HCO(3(- that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO(3(- across cells and epithelial layers. Plasma membrane anion exchange proteins (AE transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO(3(- transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl(-/HCO(3(- exchange activity in transfected HEK293 cells. Treatment of AE1-transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wild-type CAII from AE1, a loss of transport activity would be observed if binding to the AE1 C-terminus is required for transport.

  1. /sup 35/Cl and /sup 81/Br nuclear magnetic resonance studies of carbonic anhydrase

    Energy Technology Data Exchange (ETDEWEB)

    Ward, R.L.

    1979-02-01

    /sup 35/Cl NMR studies substantiated the binding of Cl/sup -/ to the Zn(II) of carbonic anhydrase. Zinc-free carbonic anhydrase was prepared and it exhibited essentially no effect on the Cl/sup -/ line width. The net Cl/sup -/ line width increased with temperature. /sup 81/Br NMR was quite similar to /sup 35/Cl in that its relaxation is dominated by quadrupolar interactions.

  2. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

    DEFF Research Database (Denmark)

    Pedersen, Daniella Bach; Koch Jensen, Peter; la Cour, Morten;

    2005-01-01

    Carbonic anhydrase inhibitors (CAIs) increase blood flow in the brain and probably also in the optic nerve and retina. Additionally they elevate the oxygen tension in the optic nerve in the pig. We propose that they also raise the oxygen tension in the retina. We studied the oxygen tension in the...... pig retina and optic nerve before and after dorzolamide injection. Also the retinal vessel diameters during carbonic anhydrase inhibition were studied....

  3. Carbonic anhydrase immobilized on hollow fiber membranes using glutaraldehyde activated chitosan for artificial lung applications

    OpenAIRE

    Kimmel, J. D.; Arazawa, D. T.; Ye, S.-H.; Shankarraman, V; Wagner, W. R.; Federspiel, W. J.

    2013-01-01

    Extracorporeal CO2 removal from circulating blood is a promising therapeutic modality for the treatment of acute respiratory failure. The enzyme carbonic anhydrase accelerates CO2 removal within gas exchange devices by locally catalyzing HCO3− into gaseous CO2 within the blood. In this work, we covalently immobilized carbonic anhydrase on the surface of polypropylene hollow fiber membranes using glutaraldehyde activated chitosan tethering to amplify the density of reactive amine functional gr...

  4. Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

    DEFF Research Database (Denmark)

    Pedersen, Daniella Bach; Koch Jensen, Peter; la Cour, Morten;

    2005-01-01

    Carbonic anhydrase inhibitors (CAIs) increase blood flow in the brain and probably also in the optic nerve and retina. Additionally they elevate the oxygen tension in the optic nerve in the pig. We propose that they also raise the oxygen tension in the retina. We studied the oxygen tension in the...... in the pig retina and optic nerve before and after dorzolamide injection. Also the retinal vessel diameters during carbonic anhydrase inhibition were studied....

  5. Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; Fisher, Gillian M; Andrews, Katherine T; Poulsen, Sally-Ann; Capasso, Clemente; Supuran, Claudiu T

    2014-09-15

    The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report. PMID:25168745

  6. Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing

    DEFF Research Database (Denmark)

    Pedersen, D B; Eysteinsson, T; Stefánsson, E;

    2004-01-01

    Prostaglandins are important in blood flow regulation. Carbon dioxide (CO(2)) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension...... (ONPO(2)), the authors investigated how indomethacin affects ONPO(2) and the ONPO(2) increases caused by CO(2) breathing and carbonic anhydrase inhibition in the pig....

  7. Capsaicin: A Potent Inhibitor of Carbonic Anhydrase Isoenzymes

    Directory of Open Access Journals (Sweden)

    Betul Arabaci

    2014-07-01

    Full Text Available Carbonic anhydrase (CA, EC 4.2.1.1 is a zinc containing metalloenzyme that catalyzes the rapid and reversible conversion of carbon dioxide (CO2 and water (H2O into a proton (H+ and bicarbonate (HCO3– ion. On the other hand, capsaicin is the main component in hot chili peppers and is used extensively used in spices, food additives and drugs; it is responsible for their spicy flavor and pungent taste. There are sixteen known CA isoforms in humans. Human CA isoenzymes I, and II (hCA I and hCA II are ubiquitous cytosolic isoforms. In this study, the inhibition properties of capsaicin against the slow cytosolic isoform hCA I, and the ubiquitous and dominant rapid cytosolic isozymes hCA II were studied. Both CA isozymes were inhibited by capsaicin in the micromolar range. This naturally bioactive compound has a Ki of 696.15 µM against hCA I, and of 208.37 µM against hCA II.

  8. Increased oxidation-related glutathionylation and carbonic anhydrase activity in endometriosis.

    Science.gov (United States)

    Andrisani, Alessandra; Donà, Gabriella; Brunati, Anna Maria; Clari, Giulio; Armanini, Decio; Ragazzi, Eugenio; Ambrosini, Guido; Bordin, Luciana

    2014-06-01

    This study examined the possible involvement of carbonic anhydrase activation in response to an endometriosis-related increase in oxidative stress. Peripheral blood samples obtained from 27 healthy controls and 30 endometriosis patients, classified as having endometriosis by histological examination of surgical specimens, were analysed by multiple immunoassay and carbonic anhydrase activity assay. Red blood cells (RBC) were analysed for glutathionylated protein (GSSP) content in the membrane, total glutathione (GSH) in the cytosol and carbonic anhydrase concentration and activity. In association with a membrane increase of GSSP and a cytosolic decrease of GSH content in endometriosis patients, carbonic anhydrase significantly increased (P < 0.0001) both monomerization and activity compared with controls. This oxidation-induced activation of carbonic anhydrase was positively and significantly correlated with the GSH content of RBC (r = 0.9735, P < 0.001) and with the amount of the 30-kDa monomer of carbonic anhydrase (r = 0.9750, P < 0.001). Because carbonic anhydrase activation is implied in many physiological and biochemical processes linked to pathologies such as glaucoma, hypertension, obesity and infections, carbonic anhydrase activity should be closely monitored in endometriosis. These data open promising working perspectives for diagnosis and treatment of endometriosis and hopefully of other oxidative stress-related diseases. Endometriosis is a chronic disease associated with infertility and local inflammatory response, which is thought to spread rapidly throughout the body as a systemic subclinical inflammation. One of the causes in the pathogenesis/evolution of endometriosis is oxidative stress, which occurs when reactive oxygen species are produced faster than the endogenous antioxidant defence systems can neutralize them. Once produced, reactive oxygen species can alter the morphological and functional properties of endothelial cells, including

  9. Carbonic anhydrase IX in early-stage non-small cell lung cancer.

    NARCIS (Netherlands)

    Kim, S.; Rabbani, Z.N.; Vollmer, R.T.; Schreiber, E.G.; Oosterwijk, E.; Dewhirst, M.W.; Vujaskovic, Z.; Kelley, M.J.

    2004-01-01

    PURPOSE: Tumor hypoxia is associated with poor prognosis and increased tumor aggressiveness. Carbonic anhydrase (CA) IX, an endogenous marker for tumor hypoxia, catalyzes the hydration of carbon dioxide into carbonic acid and contributes to the pH regulation of tumor cells. Therefore, CA IX might al

  10. Non-destructive measurement of carbonic anhydrase activity and the oxygen isotope composition of soil water

    Science.gov (United States)

    Jones, Sam; Sauze, Joana; Ogée, Jérôme; Wohl, Steven; Bosc, Alexandre; Wingate, Lisa

    2016-04-01

    Carbonic anhydrases are a group of metalloenzymes that catalyse the hydration of aqueous carbon dioxide (CO2). The expression of carbonic anhydrase by bacteria, archaea and eukarya has been linked to a variety of important biological processes including pH regulation, substrate supply and biomineralisation. As oxygen isotopes are exchanged between CO2 and water during hydration, the presence of carbonic anhydrase in plants and soil organisms also influences the oxygen isotope budget of atmospheric CO2. Leaf and soil water pools have distinct oxygen isotope compositions, owing to differences in pool sizes and evaporation rates, which are imparted on CO2during hydration. These differences in the isotopic signature of CO2 interacting with leaves and soil can be used to partition the contribution of photosynthesis and soil respiration to net terrestrial CO2 exchange. However, this relies on our knowledge of soil carbonic anhydrase activity and currently, the prevalence and function of these enzymes in soils is poorly understood. Isotopic approaches used to estimate soil carbonic anhydrase activity typically involve the inversion of models describing the oxygen isotope composition of CO2 fluxes to solve for the apparent, potentially catalysed, rate of oxygen exchange during hydration. This requires information about the composition of CO2 in isotopic equilibrium with soil water obtained from destructive, depth-resolved soil water sampling. This can represent a significant challenge in data collection given the considerable potential for spatial and temporal variability in the isotopic composition of soil water and limited a priori information with respect to the appropriate sampling resolution and depth. We investigated whether we could circumvent this requirement by constraining carbonic anhydrase activity and the composition of soil water in isotopic equilibrium with CO2 by solving simultaneously the mass balance for two soil CO2 steady states differing only in the

  11. N-Nitrosulfonamides: A new chemotype for carbonic anhydrase inhibition.

    Science.gov (United States)

    Nocentini, Alessio; Vullo, Daniela; Bartolucci, Gianluca; Supuran, Claudiu T

    2016-08-15

    A series of N(1)-substituted aromatic sulfonamides was obtained by applying a selective sulfonamide nitration synthetic strategy leading to Ar-SO2NHNO2 derivatives which were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Two human (h) hCA isoforms, the cytosolic hCA II and the transmembrane hCA IX, in addition to the fungal enzyme from Malassezia globosa, MgCA, were included in the study. Most of the new compounds reported selectively inhibited hCA IX over hCA II and at the same time showed effective MgCA inhibitory properties, with KIs ranging between 0.22 and 8.09μM. The N-nitro sulfonamides are a new chemotype with CA inhibitory effects. As hCA IX was recently validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as potential anti-dandruff agents, the N-nitro sulfonamides may be considered as interesting leads for the design of more efficient compounds targeting fungal enzymes. PMID:27290692

  12. A magnificent enzyme superfamily: carbonic anhydrases, their purification and characterization.

    Science.gov (United States)

    Ozensoy Guler, Ozen; Capasso, Clemente; Supuran, Claudiu T

    2016-10-01

    In this paper, we reviewed the purification and characterization methods of the α-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (α-, β-, γ-, δ-, ζ- and η-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of α-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the η-CAs, considered for long time an α-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. α-CAs possess important application in medicine (as many human α-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO2 capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action. PMID:26118417

  13. Carbonic anhydrase isozymes Ⅸ and Ⅻ in gastric tumors

    Institute of Scientific and Technical Information of China (English)

    Mari Leppilampi; Juha Saarnio; Tuomo J. Karttunen; Jyrki Kivel(a); Silvia Pastorekov(a); Jaromir Pastorek; Abdul Waheed; William S. Sly; Seppo Parkkila

    2003-01-01

    AIM: To systematically study the expression of carbonic anhydrase (CA) isowmes Ⅸ and Ⅻ in gastric tumors.METHODS: We analyzed a representative series of specimens from non-neoplastic gastric mucosa and from various dysplastic and neoplastic gastric lesions for the expression of CA IX and XII. Immunohistochemical staining was performed using isozyme-specific antibodies and biotinstreptavidin complex method.RESULTS: CA IX was highly expressed in the normal gastric mucosa and remained positive in many gastric tumors. In adenomas, CA IX expression significantly decreased towards the high grade dysplasia. However, the expression resumed back to the normal level in well differentiated adenocarcinomas,while it again declined in carcinomas with less differentiation.In comparison, CA Ⅻ showed no or weak immunoreaction in the normal gastric mucosa and was slightly increased in tumors.CONCLUSION: These results demonstrate that CA Ⅸexpression is sustained in several types of gastric tumors.The variations observed in the CA Ⅸ levels support the concept that gastric adenomas and carcinomas are distinct entities and do not represent progressive steps of a single pathway.

  14. Glaucoma and the applications of carbonic anhydrase inhibitors.

    Science.gov (United States)

    Scozzafava, Andrea; Supuran, Claudiu T

    2014-01-01

    Inhibition of carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the treatment of glaucoma, a disease affecting a large number of people and characterized by an elevated intraocular pressure (IOP). At least three isoforms, CA II, IV and XII are targeted by the sulfonamide inhibitors, some of which are clinically used drugs. Acetazolamide, methazolamide and dichlorophenamide are first generation CA inhibitors (CAIs) still used as systemic drugs for the management of this disease. Dorzolamide and brinzolamide represent the second generation inhibitors, being used topically, as eye drops, with less side effects compared to the first generation drugs. Third generation inhibitors have been developed by using the tail approach, but they did not reach the clinics yet. The most promising such derivatives are the sulfonamides incorporating either tails with nitric oxide releasing moieties or hybrid drugs possessing prostaglandin (PG) F agonist moieties in their molecules. Recently, the dithiocarbamates have also been described as CAIs possessing IOP lowering effects in animal models of glaucoma. CAIs are used alone or in combination with other drugs such as adrenergic agonist/antagonists, or PG analogs, being an important component of the antiglaucoma drugs armamentarium. PMID:24146387

  15. New natural product carbonic anhydrase inhibitors incorporating phenol moieties.

    Science.gov (United States)

    Karioti, Anastasia; Ceruso, Mariangela; Carta, Fabrizio; Bilia, Anna-Rita; Supuran, Claudiu T

    2015-11-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. They represent a typical example of enzyme convergent evolution, as six genetically unrelated families of such enzymes were described so far. The need to find selective CA inhibitors (CAIs) triggered the investigation of natural product libraries, which proved to be a valid source of agents with such an activity, as demonstrated for the phenols, polyamines and coumarins. Herein we report an in vitro inhibition study of human (h) CA isoforms hCAs I, II, IV, VII and XII with a panel of natural polyphenols including flavones, flavonols, flavanones, flavanols, isoflavones and depsides, some of which extracted from Quercus ilex and Salvia miltiorrhiza. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting some important isoforms over the off-target ones hCA I and II.

  16. IgM natural autoantibodies against bromelain-treated mouse red blood cells recognise carbonic anhydrase.

    Science.gov (United States)

    Jonusys, A M; Cox, K O; Steele, E J

    1991-01-01

    Carbonic anhydrase (CA) from mouse erythrocyte membranes is recognised as an autoantigen in Western blotting experiments with FUB 1, a murine IgM monoclonal antibody that binds both phosphatidylcholine and bromelain-treated mouse red blood cells (BrMRBC). Serum from mice stimulated with lipopolysaccharide (LPS-serum) also recognises CA. From SDS-PAGE, and blotting experiments with whole mouse erythrocytes, we found two closely spaced glycoprotein bands in the 30 kD region that reacted with both FUB 1 and LPS-serum. One of the molecular weight markers, bovine carbonic anhydrase which is of a molecular weight of about 30 kD, electrophoresed in the same 30 kD region also reacted with these antibodies. Carbonic anhydrases from a range of mammalian species were found to be crossreactive with FUB 1 and LPS-serum by Western blotting, whereas human glycophorin A and human asialoglycophorin were not recognised by the antibodies. FUB 1 specifically recognises both native and denatured bovine carbonic anhydrase in ELISA assays. The serological identity of the determinants of CA and BrMRBC was confirmed by specific absorption of both FUB 1 and LPS-serum with BrMRBC and normal mouse erythrocytes. We propose that a native autoantigenic epitope on erythrocytes may be revealed by the proteolytic action of bromelain and that this determinant is associated, at least in part, with carbonic anhydrase.

  17. Catecholamine-induced vasoconstriction is sensitive to carbonic anhydrase I activation

    Directory of Open Access Journals (Sweden)

    Puscas I.

    2001-01-01

    Full Text Available We studied the relationship between alpha- and beta-adrenergic agonists and the activity of carbonic anhydrase I and II in erythrocyte, clinical and vessel studies. Kinetic studies were performed. Adrenergic agonists increased erythrocyte carbonic anhydrase as follows: adrenaline by 75%, noradrenaline by 68%, isoprenaline by 55%, and orciprenaline by 62%. The kinetic data indicated a non-competitive mechanism of action. In clinical studies carbonic anhydrase I from erythrocytes increased by 87% after noradrenaline administration, by 71% after orciprenaline and by 82% after isoprenaline. The increase in carbonic anhydrase I paralleled the increase in blood pressure. Similar results were obtained in vessel studies on piglet vascular smooth muscle. We believe that adrenergic agonists may have a dual mechanism of action: the first one consists of a catecholamine action on its receptor with the formation of a stimulus-receptor complex. The second mechanism proposed completes the first one. By this second component of the mechanism, the same stimulus directly acts on the carbonic anhydrase I isozyme (that might be functionally coupled with adrenergic receptors, so that its activation ensures an adequate pH for stimulus-receptor coupling for signal transduction into the cell, resulting in vasoconstriction.

  18. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Science.gov (United States)

    Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

    2016-01-01

    Abstract Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4. PMID:27560991

  19. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets.

    Science.gov (United States)

    Supuran, Claudiu T

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO₂ hydration, with kcat values in the range of (3.4-8.3) × 10⁵ s(-1) and kcat/KM values of (4.7-8.5) × 10⁷ M(-1)·s(-1). In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2-88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets. PMID:27322334

  20. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    Directory of Open Access Journals (Sweden)

    Claudiu T. Supuran

    2016-06-01

    Full Text Available Carbonic anhydrases (CAs, EC 4.2.1.1 are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO2 hydration, with kcat values in the range of (3.4–8.3 × 105 s−1 and kcat/KM values of (4.7–8.5 × 107 M−1·s−1. In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3–90.5 nM. The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2–88.5 nM. Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets.

  1. Thermodynamics of binding of Zn2+ to carbonic anhydrase inhibitors

    Science.gov (United States)

    Remko, Milan; Garaj, Vladimír

    The Becke3LYP functional of DFT theory and the two-layered ONIOM (B3LYP/6-311+G(d,p): MNDO) method were used to characterize 46 gas-phase complexes of 34 neutral and anionic ligands (H2O, CH3OH, CH3COOH, CH3CONH2, HOSO2NH2, CO2, HSO2NH2, CH3SO2NH2, CH3C(=O)NHOH, imidazole, NH2SO2NH2, anions of 4-aminobenzenesulphonamide, saccharin, 1I9L, brinzolamide, dorzolamide, acetazolamide, further HO(-), CH3O(-), CH3COO(-), CH3CONH(-), N=N=N(-), S=C=N(-), CH3C(=O)NHO(-), HOCOO(-), imidazoleN(-), phenol-O(-), HOSO2NH(-), (-)OSO2NH(-), (-)OSO2NH2, H2NSO2NH(-), HSO2NH(-), CH3SO2NH(-), and CF3SO2NH(-), respectively) with Zn2+. Proton dissociation enthalpies and Gibbs energies of acidic inhibitors in the presence of zinc were computed. Their gas-phase acidity considerably increases upon chelation. Of the bases investigated, the weakest zinc affinity is exhibited by carbon dioxide (-313.5 kJ mol-1). Deprotonated inhibitors have higher affinities for zinc than the neutral ones. Compared to the other mono-deprotonated ligands the acetohydroxamic acid anion has the highest affinity for zinc (-1872.7 kJ mol-1). The zinc affinity of the acetazolamide anion computed using the hybrid ONIOM (B3LYP/6-311+G(d,p): MNDO) method is in very good agreement with the full DFT ones and this method can be adopted to model large complexes of inhibitors with the active site of carbonic anhydrase.

  2. A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

    OpenAIRE

    Guilloton, M B; Lamblin, A F; Kozliak, E I; Gerami-Nejad, M; Tu, C.; Silverman, D.; Anderson, P. M.; Fuchs, J A

    1993-01-01

    Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloto...

  3. Carbonic anhydrase levels and internal lacunar CO/sub 2/ concentrations in aquatic macrophytes

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, C.I.

    1979-01-01

    Carbonic anhydrase levels were examined in a variety of aquatic macrophytes from different habitats. In general, carbonic anhydrase levels increased across the habitat gradient such that activities were low in submersed aquatic macrophytes and high in emergent macrophytes with floating-leaved and free-floating plants exhibiting intermediate activities. Internal lacunar CO/sub 2/ concentrations were analyzed in relation to carbonic anhydrase activities. There was no correlation between these two parameters. Internal CO/sub 2/ concentrations ranged from low to high in submersed macrophytes, but were low in floating-leaved and emergent macrophytes. The observed internal CO/sub 2/ concentrations are discussed in relation to the individual morphologies of the plants and the environments in which they occurred.

  4. Carbonic Anhydrase II Deficiency in a Saudi Woman

    Science.gov (United States)

    Alhuzaim, Omar N; Almohareb, Ohoud M; Sherbeeni, Safiya M

    2015-01-01

    OBJECTIVE Carbonic anhydrase (CA) II deficiency is a rare autosomal recessive disorder caused by mutation in the CA II gene that leads to osteopetrosis, renal tubular acidosis (RTA), and cerebral calcification. Our aim is to present a patient with the classic triad of CA II deficiency syndrome to enhance the awareness about this rare syndrome. METHODS We describe the clinical and radiological findings of a Saudi woman patient with CA II deficiency syndrome. RESULTS A Saudi woman in her 20s presented to our hospital for evaluation of increased bone density. She was known to have delayed developmental milestone with growth retardation and poor scholastic performance. She had multiple fragile fractures started at the age of 15 involving the lower extremities. A physical examination revealed dysmorphic features and intellectual disability with intelligence quotient (IQ) of 36. The initial blood workup showed a picture of distal RTA with hypokalemia, and the radiological imaging confirmed the presence of osteopetrosis and multiple kidney stones. The combination of osteopetrosis with RTA raised the possibility of CA II deficiency. Therefore, computed tomography (CT) of the brain was done and showed intracranial calcification involving the basal ganglia. She was started on potassium chloride and sodium bicarbonate. In addition, she underwent right-sided percutaneous nephrolithotripsy. Her DNA analysis came to show a sequence variant c.232+1G>A, which was detected in both of the CA II genes (homozygous). CONCLUSION Early recognition of the disease is a key, as an early appropriate treatment institution is essential in order to prevent further complications. PMID:25674028

  5. Cadmium tolerance of carbon assimilation enzymes and chloroplast in Zn/Cd hyperaccumulator Picris divaricata.

    Science.gov (United States)

    Ying, Rong-Rong; Qiu, Rong-Liang; Tang, Ye-Tao; Hu, Peng-Jie; Qiu, Hao; Chen, Hong-Ru; Shi, Tai-Hong; Morel, Jean-Louis

    2010-01-15

    To better understand the photosynthesis under stress, the effect of cadmium on carbon assimilation and chloroplast ultrastructure of a newly found Zn/Cd hyperaccumulator Picris divaricata in China was investigated in solution culture. The shoot and root Cd concentrations increased with increase in Cd supply, reaching maxima of 1109 and 5604mgkg(-1) dry weight at 75microM Cd, respectively. As Cd supply to P. divaricata increased, the shoot and root dry weight, leaf water content (except 75microM Cd), concentrations of chlorophyll a and b, chlorophyll a/b ratio and the concentration of carotenoids were not depressed at high Cd. However, the stomatal conductance, transpiration rate, net photosynthetic rate and intercellular CO(2) concentration were significantly affected when the Cd concentration reached 10, 10, 25 and 75microM, respectively. Meanwhile, carbonic anhydrase (CA; EC 4.2.1.1) activity and Rubisco (EC 4.1.1.39) content reached maxima in the presence of 50 and 5microM Cd, respectively. In addition, CA activity correlated positively with shoot Cd in plants treated with Cd at a range of 0-50microM. Moreover, the activities of NADP(+)-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13), Rubisco and fructose-1, 6-bisphosphatase (EC 3.1.3.11) were not significantly suppressed by increased Cd supply. Although the mesophyll cell size was reduced, chloroplast ultrastructure remained intact at the highest Cd treatment. Our finding revealed that P. divaricata chloroplast and the enzymes of carbon assimilation tolerate high levels of Cd, demonstrating its potential in possible application in phytoremediation. PMID:19683362

  6. Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

    OpenAIRE

    Stams, T.; Y. Chen; Boriack-Sjodin, P. A.; Hurt, J. D.; Liao, J; May, J. A.; Dean, T.; Laipis, P; Silverman, D. N.; Christianson, D. W.

    1998-01-01

    Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some...

  7. Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

    Energy Technology Data Exchange (ETDEWEB)

    Cannon, Gordon C.; Heinhorst, Sabine; Kerfeld, Cheryl A.

    2010-06-23

    Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO2 concentrations by employing a CO2-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO2-fixing enzyme, RubisCO. The efficiency of CO2 fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO2. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.

  8. Human carbonic anhydrase II as a host for piano-stool complexes bearing a sulfonamide anchor.

    Science.gov (United States)

    Monnard, Fabien W; Heinisch, Tillmann; Nogueira, Elisa S; Schirmer, Tilman; Ward, Thomas R

    2011-08-01

    d(6)-piano-stool complexes bearing an arylsulfonamide anchor display sub-micromolar affinity towards human Carbonic Anhydrase II (hCA II). The 1.3 Å resolution X-ray crystal structure of [(η(6)-C(6)Me(6))Ru(bispy 3)Cl](+)⊂ hCA II highlights the nature of the host-guest interactions. PMID:21706094

  9. Variable involvement of the perivascular retinal tissue in carbonic anhydrase inhibitor induced relaxation of porcine retinal arterioles in vitro

    DEFF Research Database (Denmark)

    Kehler, Anne Katrine; Holmgaard, Kim; Hessellund, Anders;

    2007-01-01

    PURPOSE: Inhibition of carbonic anhydrase in the eye is an important treatment modality for reducing the intraocular pressure in glaucoma. However, evidence suggests that carbonic anhydrase inhibition also exerts a relaxing effect on the vessels in the optic nerve, and it has been suggested......, and the vasodilating effect of acetazolamide almost disappeared. CONCLUSIONS: A further elucidation of the mechanisms of action of carbonic anhydrase-induced dilation of retinal arterioles may contribute to a better understanding of the regulation of retinal blood flow. The perivascular retinal tissue may play...... a significant role in diameter control of retinal arterioles. Udgivelsesdato: 2007-Oct...

  10. Expression and Activity of Carbonic Anhydrase IX Is Associated With Metabolic Dysfunction in MDA-MB-231 Breast Cancer Cells

    OpenAIRE

    Ying LI; Wang, Hai; Oosterwijk, Egbert; Tu, Chingkuang; Shiverick, Kathleen T.; Silverman, David N.; Frost, Susan C.

    2009-01-01

    The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a “triple-negative,” basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density-and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracel...

  11. Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

    Science.gov (United States)

    Kozliak, E I; Guilloton, M B; Gerami-Nejad, M; Fuchs, J A; Anderson, P M

    1994-09-01

    Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.

  12. Targeting carbonic anhydrase to treat diabetic retinopathy: Emerging evidences and encouraging results

    Energy Technology Data Exchange (ETDEWEB)

    Weiwei, Zhang [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China); Hu, Renming, E-mail: taylorzww@gmail.com [Department of Endocrinology and Metabolism, HuaShan Hospital, Institute of Endocrinology and Diabetology, Shanghai Medical College, Fudan University, No. 12 Wulumuqi Road, Shanghai 200040 (China)

    2009-12-18

    Diabetic retinopathy (DR) is the leading cause of vision loss among working-age populations in developed countries. Current treatment options are limited to tight glycemic, blood pressure control and destructive laser surgery. Carbonic anhydrases (CAs) are a group of enzymes involving in the rapid conversion of carbon dioxide to bicarbonate and protons. Emerging evidences reveal CA inhibitors hold the promise for the treatment of DR. This article summarizes encouraging results from clinical and animal studies, and reviews the possible mechanisms.

  13. Targeting carbonic anhydrase to treat diabetic retinopathy: Emerging evidences and encouraging results

    International Nuclear Information System (INIS)

    Diabetic retinopathy (DR) is the leading cause of vision loss among working-age populations in developed countries. Current treatment options are limited to tight glycemic, blood pressure control and destructive laser surgery. Carbonic anhydrases (CAs) are a group of enzymes involving in the rapid conversion of carbon dioxide to bicarbonate and protons. Emerging evidences reveal CA inhibitors hold the promise for the treatment of DR. This article summarizes encouraging results from clinical and animal studies, and reviews the possible mechanisms.

  14. A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli.

    Science.gov (United States)

    Guilloton, M B; Lamblin, A F; Kozliak, E I; Gerami-Nejad, M; Tu, C; Silverman, D; Anderson, P M; Fuchs, J A

    1993-03-01

    Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and

  15. Carbonic anhydrase mediated carbon dioxide sequestration: promises, challenges and future prospects.

    Science.gov (United States)

    Yadav, Raju R; Krishnamurthi, Kannan; Mudliar, Sandeep N; Devi, S Saravana; Naoghare, Pravin K; Bafana, Amit; Chakrabarti, Tapan

    2014-06-01

    Anthropogenic activities have substantially increased the level of greenhouse gases (GHGs) in the atmosphere and are contributing significantly to the global warming. Carbon dioxide (CO2 ) is one of the major GHGs which plays a key role in the climate change. Various approaches and methodologies are under investigation to address CO2 capture and sequestration worldwide. Carbonic anhydrase (CA) mediated CO2 sequestration is one of the promising options. Therefore, the present review elaborates recent developments in CA, its immobilization and bioreactor methodologies towards CO2 sequestration using the CA enzyme. The promises and challenges associated with the efficient utilization of CA for CO2 sequestration and scale up from flask to lab-scale bioreactor are critically discussed. Finally, the current review also recommends the possible future needs and directions to utilize CA for CO2 sequestration. PMID:24740638

  16. Diminished Cellular Immune Response to Carbonic Anhydrase II in Patients with Sjogren's Syndrome and Idiopathic Chronic Pancreatitis

    Directory of Open Access Journals (Sweden)

    Onishi S

    2004-07-01

    Full Text Available CONTEXT: A serum antibody to carbonic anhydrase II has been reported in patients with Sjögren’s syndrome and idiopathic chronic pancreatitis. OBJECTIVE: To evaluate cellular immune response to carbonic anhydrase II in patients with Sjögren’s syndrome and idiopathic chronic pancreatitis. PATIENTS: Idiopathic chronic pancreatitis (n=23, Sjögren’s syndrome (n=12, alcoholic chronic pancreatitis (n=3 and normal controls (n=13. MAIN OUTCOME MEASURES: Proliferation assay of peripheral blood mononuclear cells. RESULTS: Notable increased proliferation of the mononuclear cells upon stimulation with carbonic anhydrase II was observed in 2 patients with idiopathic chronic pancreatitis (9% and 2 patients with Sjögren’s syndrome (17% but not in patients with alcoholic chronic pancreatitis nor in normal controls. Among the four study groups, there was no significant difference in the prevalence rate of the positive proliferative responses (P=0.444. CONCLUSION: Carbonic anhydrase II may not be a major target antigen for the immunological process in the pathogenesis of Sjögren’s syndrome and idiopathic chronic pancreatitis. Serum antibody to carbonic anhydrase II may be detected in these patients as a consequence of the immune reaction against other antigens which mimic carbonic anhydrase II.

  17. [Targeting of type IV carbonic anhydrases in Capan-1 human pancreatic duct cells is concomitant of the polarization].

    Science.gov (United States)

    Mairal, A; Fanjul, M; Hollande, E

    1996-01-01

    Carbonic anhydrases II and IV play an essential role in the synthesis and secretion of HCO3- ions in pancreatic duct cells. Secretion of these ions is regulated by the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. In the present study, the expression of carbonic anhydrases IV and their targeting to plasma membranes were examined during the growth of human pancreatic duct cells in vitro. Human cancerous pancreatic duct cells of Capan-1 cell line which polarize during their growth were used. We show that: a) these cells express carbonic anhydrases IV continuously during growth in culture, and the expression depends on the stage of growth and the conformation of the cells; b) carbonic anhydrases IV are seen in the cytoplasm in non-polarized cells, but become progressively anchored to plasma membranes as the cells polarize, being targeted to the apical membranes of polarized cells; c) the subcellular distribution of carbonic anhydrases IV indicates that these enzymes are synthetized in rough endoplasmic reticulum and then transported towards the plasma membrane using the classical secretory pathway through the Golgi apparatus. The results indicated that targeting of carbonic anhydrases IV in Capan-1 cells is linked to cellular polarization. PMID:8881572

  18. Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides.

    Directory of Open Access Journals (Sweden)

    Joyanta K Modak

    Full Text Available Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA, an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

  19. Carbonic anhydrase activity in the red blood cells of sea level and high altitude natives.

    Science.gov (United States)

    Gamboa, J; Caceda, R; Gamboa, A; Monge-C, C

    2000-01-01

    Red blood cell carbonic anhydrase (CA) activity has not been studied in high altitude natives. Because CA is an intraerythocytic enzyme and high altitude natives are polycythemic, it is important to know if the activity of CA per red cell volume is different from that of their sea level counterparts. Blood was collected from healthy subjects living in Lima (150m) and from twelve subjects from Cerro de Pasco (4330m), and hematocrit and carbonic anhydrase activity were measured. As expected, the high altitude natives had significantly higher hematocrits than the sea level controls (p = 0.0002). No difference in the CA activity per milliliter of red cells was found between the two populations. There was no correlation between the hematocrit and CA activity.

  20. [Mode of action, clinical profile and relevance of carbonic anhydrase inhibitors in glaucoma therapy].

    Science.gov (United States)

    Eichhorn, M

    2013-02-01

    Since their introduction the local carbonic anhydrase inhibitors (CAH) dorzolamide and brinzolamide have become well established in the drug therapy of glaucoma. They lower intraocular pressure (IOP) by blocking specifically carbonic anhydrase in the ciliary epithelium and thereby the secretion of aqueous humor. The IOP lowering effect is comparable with that of beta-blockers, but less than that of prostaglandin agonists. Because of their specific mode of action they produce an additive pressure lowering effect with any other glaucoma drug. Therefore they are ideal for being combined with other drugs. In addition, CAH may improve perfusion of the posterior eye. Preliminary results in glaucoma patients under dorzolamide therapy suggesting a reduction in the risk of progression due to enhanced blood flow need further confirmation. PMID:23430679

  1. Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations.

    OpenAIRE

    Carta, Fabrizio; Aggarwal, Mayank; Maresca, Alfonso; Scozzafava, Andrea; McKenna, Robert; Supuran, Claudiu T.

    2012-01-01

    The zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) is inhibited by several classes of zinc-binders (sulfonamides, sulfamates, and sulfamides) as well as by compounds which do not interact with the metal ion (phenols, polyamines and coumarins). Here we report a new class of potent CA inhibitors which bind the zinc ion: the dithiocarbamates (DTCs). They coordinate to the zinc ion from the enzyme active site in monodentate manner and establish many favorable interactions with amino acid residue...

  2. Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

    OpenAIRE

    Reibring, Claes-Göran; El Shahawy, Maha; Hallberg, Kristina; Kannius-Janson, Marie; Nilsson, Jeanette; Parkkila, Seppo; Sly, William S; Waheed, Abdul; Linde, Anders; Gritli-Linde, Amel

    2014-01-01

    Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution pa...

  3. Toxic Epidermal Necrolysis Induced by the Topical Carbonic Anhydrase Inhibitors Brinzolamide and Dorzolamide

    OpenAIRE

    Chun, Ji Sun; Yun, Sook Jung; Lee, Jee Bum; Kim, Seong Jin; Won, Young Ho; Lee, Seung Chul

    2008-01-01

    Brinzolamide and dorzolamide are highly specific topical carbonic anhydrase inhibitors (CAIs). They lower intraocular pressure (IOP) by reducing the rate of aqueous humour formation without serious side effects. Although systemic CAIs are the most potent medications for lowering intraocular pressure for conditions with ocular hypertension, many cases with adverse systemic reactions have been reported, including Stevens-Johnson syndrome (SJS) and Toxic epidermal necrolysis (TEN). Here, we repo...

  4. Strong topical steroid, NSAID, and carbonic anhydrase inhibitor cocktail for treatment of cystoid macular edema

    Directory of Open Access Journals (Sweden)

    Asahi MG

    2015-12-01

    Full Text Available Masumi G Asahi, Gabriela L Bobarnac Dogaru, Spencer M Onishi, Ron P GallemoreRetina Macula Institute, Torrance, CA, USA Purpose: To report the combination cocktail of strong steroid, non-steroidal anti-inflammatory drug (NSAID, and carbonic anhydrase inhibitor drops for treatment of cystoid macular edema. Methods: This is a retrospective case series of patients with cystoid macular edema managed with a topical combination of strong steroid (difluprednate, NSAID, and carbonic anhydrase inhibitor drops. The patients were followed with optical coherence tomography and fluorescein angiography. Results: In our six cases, resolution of the cystic edema with improvement in visual acuity was achieved with the use of a combination cocktail of drops. Leakage on fluorescein angiography and cystic edema on optical coherence tomography both responded to treatment with the topical cocktail of drops. Conclusion: A topical cocktail of strong steroid, NSAID, and carbonic anhydrase inhibitor drops are effective for managing cystoid macular edema. Further studies comparing this combination with more invasive treatments should be undertaken to determine the efficacy of this cocktail over other treatment options. Keywords: birdshot chorioretinopathy, diabetic macular edema, retinal vein occlusion

  5. Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells

    KAUST Repository

    Hu, Honghong

    2009-12-13

    The continuing rise in atmospheric CO2 causes stomatal pores in leaves to close and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress. However, the CO2-binding proteins that control this response remain unknown. Moreover, which cell type responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate. We demonstrate that Arabidopsis thaliana double-mutant plants in the beta-carbonic anhydrases betaCA1 and betaCA4 show impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses. betaCA-mediated CO2-triggered stomatal movements are not, in first-order, linked to whole leaf photosynthesis and can function in guard cells. Furthermore, guard cell betaca-overexpressing plants exhibit instantaneous enhanced water use efficiency. Guard cell expression of mammalian alphaCAII complements the reduced sensitivity of ca1 ca4 plants, showing that carbonic anhydrase-mediated catalysis is an important mechanism for betaCA-mediated CO2-induced stomatal closure and patch clamp analyses indicate that CO2/HCO3- transfers the signal to anion channel regulation. These findings, together with ht1-2 (ref. 9) epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway, which controls gas-exchange between plants and the atmosphere.

  6. Screening and docking studies of natural phenolic inhibitors of carbonic anhydrase

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Carbonic anhydrase Ⅱ (CAⅡ) is an important enzyme complex with Zn2+,which is involved in many physiological and pathological processes, such as calcification, glaucoma and tumorigenicity. In order to search for novel inhibitors of CAⅡ, inhibition assay of carbonic anhydrase Ⅱ was performed, by which seven natural phenolic compounds, including four phenolics (grifolin, 4-O-methyl-grifolic acid, grifolic acid, and isovanillic acid) and three flavones (eriodictyol, quercetin and puerin A), showed in-hibitory activities against CAⅡ with IC50s in the range of 6.37-71.73 μmol/L. Grifolic acid is the most active one with IC50 of 6.37 μmol/L. These seven phenolic compounds were proved to be novel natural carbonic anhydrase Ⅱ inhibitors, which were obtained in flexible docking study with GOLD 3.0 soft-ware. Results indicated that the aliphatic chain and polar groups of hydroxyl and carboxyl are impor-tant to their inhibitory activities, providing a new insight into study on CA Ⅱ potent inhibitors.

  7. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    Science.gov (United States)

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-01-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions. PMID:27385052

  8. Engineering de novo disulfide bond in bacterial α-type carbonic anhydrase for thermostable carbon sequestration

    Science.gov (United States)

    Jo, Byung Hoon; Park, Tae Yoon; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je; Cha, Hyung Joon

    2016-07-01

    Exploiting carbonic anhydrase (CA), an enzyme that rapidly catalyzes carbon dioxide hydration, is an attractive biomimetic route for carbon sequestration due to its environmental compatibility and potential economic viability. However, the industrial applications of CA are strongly hampered by the unstable nature of enzymes. In this work, we introduced in silico designed, de novo disulfide bond in a bacterial α-type CA to enhance thermostability. Three variants were selected and expressed in Escherichia coli with an additional disulfide bridge. One of the variants showed great enhancement in terms of both kinetic and thermodynamic stabilities. This improvement could be attributed to the loss of conformational entropy of the unfolded state, showing increased rigidity. The variant showed an upward-shifted optimal temperature and appeared to be thermoactivated, which compensated for the lowered activity at 25 °C. Collectively, the variant constructed by the rapid and effective de novo disulfide engineering can be used as an efficient biocatalyst for carbon sequestration under high temperature conditions.

  9. Dithiocarbamates Strongly Inhibit Carbonic Anhydrases and Show Antiglaucoma Action in Vivo

    OpenAIRE

    Carta, Fabrizio; Aggarwal, Mayank; Maresca, Alfonso; Scozzafava, Andrea; McKenna, Robert; Masini, Emanuela; Supuran, Claudiu T.

    2012-01-01

    A series of dithiocarbamates was prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of 4 human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. X-ray crystal structure of hCA II adduct with morpholine dithiocarbamate ...

  10. Oxygen-18 incorporation into malic acid during nocturnal carbon dioxide fixation in crassulacean acid metabolism plants: a new approach to estimating in vivo carbonic anhydrase activity

    Energy Technology Data Exchange (ETDEWEB)

    Holtum, J.A.M.; Summons, R.; Roeske, C.A.; Comins, H.N.; O' Leary, M.H.

    1984-01-01

    Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with TC YO2, then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the ( TC) malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance or by mass spectrometry. Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum, 40 for Kalanchoee daigremontiana, and 100 or greater for Bryophyllum tubiflorum, Kalanchoee serrata, and Kalanchoae tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature. 37 references, 2 figures, 8 tables.

  11. Evidence that an internal carbonic anhydrase is present in 5% CO2-grown and air-grown Chlamydomonas

    International Nuclear Information System (INIS)

    Inorganic carbon (C/sub i/) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO2. Both air-grown cells, that have a CO2 concentrating system, and 5% CO2-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C/sub i/) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO2-grown cells also accumulated some C/sub i/, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO2 fixation by high CO2-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO2-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase

  12. Carbon-13 nuclear magnetic resonance as a probe of side chain orientation and mobility in carboxymethylated human carbonic anhydrase B

    NARCIS (Netherlands)

    Schoot Uiterkamp, Antonius J.M.; Armitage, Ian M.; Prestegard, James H.; Slomski, John; Coleman, Joseph E.

    1978-01-01

    13C NMR spectra of [1-13C]- and [2-13C]carboxymethyl His-200 human carbonic anhydrase B have been obtained as a function of pH and in the presence and absence of the active site Zn(II) or Cd(II) ion. Chemical shifts of the 1-13C show that the carboxyl is sensitive to two ionization processes, with a

  13. Cadmium-Containing Carbonic Anhydrase CDCA1 in Marine Diatom Thalassiosira weissflogii

    Directory of Open Access Journals (Sweden)

    Vincenzo Alterio

    2015-03-01

    Full Text Available The Carbon Concentration Mechanism (CCM allows phytoplakton species to accumulate the dissolved inorganic carbon (DIC necessary for an efficient photosynthesis even under carbon dioxide limitation. In this mechanism of primary importance for diatoms, a key role is played by carbonic anhydrase (CA enzymes which catalyze the reversible hydration of CO2, thus taking part in the acquisition of inorganic carbon for photosynthesis. A novel CA, named CDCA1, has been recently discovered in the marine diatom Thalassiosira weissflogii. CDCA1 is a cambialistic enzyme since it naturally uses Cd2+ as catalytic metal ion, but if necessary can spontaneously exchange Cd2+ to Zn2+. Here, the biochemical and structural features of CDCA1 enzyme will be presented together with its putative biotechnological applications for the detection of metal ions in seawaters.

  14. Expression and activity of carbonic anhydrase IX is associated with metabolic dysfunction in MDA-MB-231 breast cancer cells.

    NARCIS (Netherlands)

    Li, Ying; Wang, H.; Oosterwijk, E.; Tu, C.; Shiverick, K.T.; Silverman, D.N.; Frost, S.C.

    2009-01-01

    The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a "triple-negative," basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydra

  15. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

    Directory of Open Access Journals (Sweden)

    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  16. Relationship among Photosys- tem Ⅱ carbonic anhydrase, extrinsic polypeptides and manganese cluster

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Effects of Photosystem Ⅱ (PS Ⅱ) extrinsic poly- peptides of oxygen-evolving complex and manganese clusters on PSⅡ carbonic anhydrase (CA) were studied with spinach PSⅡ membranes. The result supported that membrane-bound CA is located in the donor side of PSⅡ. The extrinsic polypeptides played an important role of maintaining CA activity. After removing manganese clusters, oxygen evolution activity was inhibited, but PSⅡ-CA activity was unchanged. It was concluded that CA activity is independent of the presence of manganese clusters, and was not directly correlated with oxygen evolution activity.

  17. Effects of carbonic anhydrase inhibition on ventilation-perfusion matching in the dog lung.

    OpenAIRE

    Swenson, E.R.; Robertson, H T; Hlastala, M P

    1993-01-01

    Lung carbonic anhydrase (CA) permits rapid pH responses when changes in regional ventilation or perfusion alter airway and alveolar PCO2. These pH changes affect airway and vascular resistances and lung compliance to optimize the balance of regional ventilation (VA) and perfusion (Q) in the lung. To test the hypothesis that these or other CA-dependent mechanisms contribute to VA/Q matching, we administered acetazolamide (25 mg/kg intravenously) to six anesthetized and paralyzed dogs and measu...

  18. Carbonic Anhydrase as Pollution Biomarker: An Ancient Enzyme with a New Use

    Directory of Open Access Journals (Sweden)

    Trifone Schettino

    2012-11-01

    Full Text Available The measurement of cellular and sub-cellular responses to chemical contaminants (referred to as biomarkers in living organisms represents a recent tool in environmental monitoring. The review focuses on carbonic anhydrase, a ubiquitous metalloenzyme which plays key roles in a wide variety of physiological processes involving CO2 and HCO3−. In the last decade a number of studies have demonstrated the sensitivity of this enzyme to pollutants such as heavy metals and organic chemicals in both humans and wildlife. The review analyses these studies and discusses the potentiality of this enzyme as novel biomarker in environmental monitoring and assessment.

  19. Design, synthesis, and evaluation of NO-donor containing carbonic anhydrase inhibitors to lower intraocular pressure.

    Science.gov (United States)

    Huang, Qinhua; Rui, Eugene Y; Cobbs, Morena; Dinh, Dac M; Gukasyan, Hovhannes J; Lafontaine, Jennifer A; Mehta, Saurabh; Patterson, Brian D; Rewolinski, David A; Richardson, Paul F; Edwards, Martin P

    2015-03-26

    The antiglaucoma drugs dorzolamide (1) and brinzolamide (2) lower intraocular pressure (IOP) by inhibiting the carbonic anhydrase (CA) enzyme to reduce aqueous humor production. The introduction of a nitric oxide (NO) donor into the alkyl side chain of dorzolamide (1) and brinzolamide (2) has led to the discovery of NO-dorzolamide 3a and NO-brinzolamide 4a, which could lower IOP through two mechanisms: CA inhibition to decrease aqueous humor secretion (reduce inflow) and NO release to increase aqueous humor drainage (increase outflow). Compounds 3a and 4a have shown improved efficacy of lowering IOP in both rabbits and monkeys compared to brinzolamide (2). PMID:25728019

  20. Carbon dioxide capture using Escherichia coli expressing carbonic anhydrase in a foam bioreactor.

    Science.gov (United States)

    Watson, Stuart K; Han, Zhenlin; Su, Wei Wen; Deshusses, Marc A; Kan, Eunsung

    2016-12-01

    The present study reports CO2 capture and conversion to bicarbonate using Escherichia coli expressing carbonic anhydrase (CA) on its cell surface in a novel foam bioreactor. The very large gas-liquid interfacial area in the foam bioreactor promoted rapid CO2 absorption while the CO2 in the aqueous phase was subsequently converted to bicarbonate ions by the CA. CO2 gas removal in air was investigated at various conditions such as gas velocity, cell density and CO2 inlet concentration. Regimes for kinetic and mass transfer limitations were defined. Very high removal rates of CO2 were observed: 9570 g CO2 m(-3) bioreactor h(-1) and a CO2 removal efficiency of 93% at 4% inlet CO2 when the gas retention time was 24 s, and cell concentration was 4 gdw L(-1). These performances are superior to earlier reports of experimental bioreactors using CA for CO2 capture. Overall, this bioreactor system has significant potential as an alternative CO2 capture technology.

  1. Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-{gamma}2

    Energy Technology Data Exchange (ETDEWEB)

    Mitterberger, Maria C. [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria); Kim, Geumsoo [Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012 (United States); Rostek, Ursula [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria); Levine, Rodney L. [Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012 (United States); Zwerschke, Werner, E-mail: werner.zwerschke@oeaw.ac.at [Cell Metabolism and Differentiation Research Group, Institute for Biomedical Aging Research of the Austrian Academy of Sciences, 6020 Innsbruck (Austria)

    2012-05-01

    Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO{sub 2} have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII{sup -/-} MEFs compared with CAIII{sup +/+} cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-{gamma}2 (PPAR{gamma}2) and CCAAT/enhancer binding protein-{alpha}. We found a considerable (approximately 1000-fold) increase in the PPAR{gamma}2 expression in the CAIII{sup -/-} MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPAR{gamma}2 and FABP4. When both CAIII and PPAR{gamma}2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPAR{gamma}2 gene expression. -- Highlights: Black-Right-Pointing-Pointer We discover a novel function of Carbonic anhydrase III (CAIII). Black-Right-Pointing-Pointer We show that CAIII is a regulator of adipogenesis. Black-Right-Pointing-Pointer We demonstrate that CAIII acts at the level of PPAR{gamma}2 gene expression. Black-Right-Pointing-Pointer Our data contribute to a better understanding of the role of CAIII in fat tissue.

  2. Kinetics of Formation of Cobalt(II)- and Nickel(II) Carbonic Anhydrase.

    Science.gov (United States)

    McQuate, Robert S.; Reardon, John E.

    1978-01-01

    Discusses the kinetic behavior associated with the interaction of metal ions with apocarbonic anhydrase, focusing on the formation of two metallocarbonic anhydrase--the biochemically active Co(II) and the inactive Ni(II)derivatives. (GA)

  3. Zinc Transfer Kinetics of Metallothioneins and Their Fragmentswith Apo-carbonic Anhydrase

    Institute of Scientific and Technical Information of China (English)

    HUANG, Zhong-Xian; LIU, Fang; ZHENG, Qi; YU, Wen-Hao

    2001-01-01

    Tne zinc transfer reactions from Zn7-MT-I, Zn7-MT-Ⅱ, Zn4α fragment (MT-I) and Zn4-α fragment (MT-Ⅱ) to apo-carbonic anhydrase have been studied. In each reaction, no more than one zinc ion per molecule is involved in metal transfer.Zn7-MT-I and Zn7-MT-Ⅱ donate zinc to apo-carbonic anhydrase and de novo constitute it at a comparable efficiency,while Zn7-MT-Ⅱ exhibits a little faster rate. Surprisingiy,Zinc is released from Zn4-α fragment (MT-Ⅱ) with a much faster rate than from Zn4-α fragment (MT-I), whose rate is close to that of Zn7-MT-I. The reason for the difference is still unknown. Introducing complex compounds into this system may give rise to an effect on the reaction. The transfer from Zn7-MT-Ⅱ in the presence of reduced glutathione shows little difference compare to the control, suggesting that the reduced glutathione is not involved in zinc transfer process. However,glutathione disulfide does accelerate this zinc transfer reaction remarkably, indicating that the oxidative factors contribute to zinc rlease from metallothioneins.

  4. New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

    Science.gov (United States)

    Rogez-Florent, Tiphaine; Meignan, Samuel; Foulon, Catherine; Six, Perrine; Gros, Abigaëlle; Bal-Mahieu, Christine; Supuran, Claudiu T; Scozzafava, Andrea; Frédérick, Raphaël; Masereel, Bernard; Depreux, Patrick; Lansiaux, Amélie; Goossens, Jean-François; Gluszok, Sébastien; Goossens, Laurence

    2013-03-15

    Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1μM CA IX inhibitor. PMID:23168081

  5. Catalase, carbonic anhydrase and xanthine oxidase activities in patients with mycosis fungoides.

    Science.gov (United States)

    Cengiz, Fatma Pelin; Beyaztas, Serap; Gokce, Basak; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-04-01

    Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma. In several studies the relationship between catalase (CAT), human cytosolic carbonic anhydrases (CA; hCA-I and hCA-II) and xanthine oxidase (XO) enzyme activities have been investigated in various types of cancers but carbonic anhydrase, catalase and xanthine oxidase activities in patients with MF have not been previously reported. Therefore, in this preliminary study we aim to investigate CAT, CA and XO activities in patients with MF. This study enrolled 32 patients with MF and 26 healthy controls. According to the results, CA and CAT activities were significantly lower in patients with mycosis fungoides than controls (p < 0.001) (p < 0.001). There was no significant difference in XO activity between patient and control group (p = 0.601). Within these findings, we believe these enzyme activity levels might be a potentially important finding as an additional diagnostic biochemical tool for MF.

  6. Optimizing lutetium 177-anti-carbonic anhydrase IX radioimmunotherapy in an intraperitoneal clear cell renal cell carcinoma xenograft model

    NARCIS (Netherlands)

    Muselaers, C.H.J.; Oosterwijk, E.; Bos, D.L.; Oyen, W.J.G.; Mulders, P.F.A.; Boerman, O.C.

    2014-01-01

    A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 (177Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT st

  7. Evolution of the mammary capillary network and carbonic anhydrase activity throughout lactation and during somatotropin treatment in goats

    DEFF Research Database (Denmark)

    Nielsen, Mette Benedicte Olaf; Cvek, Katarina; Dahlborn, Kristina

    2010-01-01

    During the normal course of lactation, mammary metabolic activity and blood flow are closely correlated. Six lactating goats were used in this experiment to test the hypothesis that the capillary network and the capillary enzyme, carbonic anhydrase (CA; EC 4.2.1.1) are important regulatory factors...

  8. Metabolic Effect of Estrogen Receptor Agonists on Breast Cancer Cells in the Presence or Absence of Carbonic Anhydrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Anissa Belkaid

    2016-05-01

    Full Text Available Metabolic shift is one of the major hallmarks of cancer development. Estrogen receptor (ER activity has a profound effect on breast cancer cell growth through a number of metabolic changes driven by its effect on transcription of several enzymes, including carbonic anhydrases, Stearoyl-CoA desaturase-1, and oncogenes including HER2. Thus, estrogen receptor activators can be expected to lead to the modulation of cell metabolism in estrogen receptor positive cells. In this work we have investigated the effect of 17β-estradiol, an ER activator, and ferulic acid, a carbonic anhydrase inhibitor, as well as ER activator, in the absence and in the presence of the carbonic anhydrase inhibitor acetazolamide on the metabolism of MCF7 cells and MCF7 cells, stably transfected to express HER2 (MCF7HER2. Metabolic profiles were studied using 1D and 2D metabolomic Nuclear Magnetic Resonance (NMR experiments, combined with the identification and quantification of metabolites, and the annotation of the results in the context of biochemical pathways. Overall changes in hydrophilic metabolites were largest following treatment of MCF7 and MC7HER2 cells with 17β-estradiol. However, the carbonic anhydrase inhibitor acetazolamide had the largest effect on the profile of lipophilic metabolites.

  9. Oxygen-18 incorporation into malic acid during nocturnal carbon dioxide fixation in crassulacean acid metabolism plants. A new approach to estimating in vivo carbonic anhydrase activity.

    Science.gov (United States)

    Holtum, J A; Summons, R; Roeske, C A; Comins, H N; O'Leary, M H

    1984-06-10

    Crassulacean acid metabolism (CAM) plants fix carbon dioxide at night by the carboxylation of phosphoenolpyruvate. If CO2 fixation is conducted with 13C18O2 , then in the absence of carbonic anhydrase, the malate formed by dark CO2 fixation should also contain high levels of carbon-13 and oxygen-18. Conversely, if carbonic anhydrase is present and highly active, oxygen exchange between CO2 and cellular H2O will occur more rapidly than carboxylation, and the [13C] malate formed will contain little or no oxygen-18 above the natural abundance level. The presence of oxygen-18 in these molecules can be detected either by nuclear magnetic resonance (using the oxygen-18 effect on the carbon-13 chemical shift of the carboxyl carbon) or by mass spectrometry (comparing the ions at three and five units above the molecular weight with that one unit above). Studies of phosphoenolpyruvate carboxylase in the presence and absence of carbonic anhydrase in vitro confirm the validity of the method. When CAM plants are studied by this method, we find that most species show incorporation of a significant amount of oxygen-18. Comparison of these results with results of isotope fractionation and gas exchange studies permits calculation of the in vivo activity of carbonic anhydrase toward HCO-3 compared with that of phosphoenolpyruvate carboxylase. The ratio (carbonic anhydrase activity/phosphoenolpyruvate carboxylase activity) is species dependent and varies from a low of about 7 for Ananas comosus to values near 20 for Hoya carnosa and Bryophyllum pinnatum , 40 for Kalancho ë daigremontiana , and 100 or greater for Bryophyllum tubiflorum , Kalancho ë serrata, and Kalancho ë tomentosa. Carbonic anhydrase activity increases relative to phosphoenolpyruvate carboxylase activity at higher temperature. PMID:6427227

  10. Xanthates and trithiocarbonates strongly inhibit carbonic anhydrases and show antiglaucoma effects in vivo.

    Science.gov (United States)

    Carta, Fabrizio; Akdemir, Atilla; Scozzafava, Andrea; Masini, Emanuela; Supuran, Claudiu T

    2013-06-13

    Dithiocarbamates (DTCs) were recently discovered as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. A series of xanthates and a trithiocarbonate, structurally related to the DTCs, were prepared by reaction of alcohols/thiols with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms, hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar xanthate/trithiocarbonate inhibitors targeting these CAs were detected. A docking study of some xanthates within the CA II active site showed that these compounds bind in a similar manner with the dithiocarbamates, coordinating monodentately to the Zn(II) ion from the enzyme active site. Several xanthates showed potent intraocular pressure lowering activity in two animal models of glaucoma via the topical administration. Xanthates and thioxanthates represent two novel, promising classes of CA inhibitors. PMID:23647428

  11. Carbonic Anhydrase and Zinc in Plant Physiology Anhidrasa Carbónica y Zinc en Fisiología Vegetal

    Directory of Open Access Journals (Sweden)

    Dalila Jacqueline Escudero-Almanza

    2012-03-01

    Full Text Available Carbonic anhydrase (CA (EC: 2.4.1.1 catalyzes the rapid conversion of carbon dioxide plus water into a proton and the bicarbonate ion (HCO3- that can be found in prokaryotes and higher organisms; it is represented by four different families. Carbonic anhydrase is a metalloenzyme that requires Zn as a cofactor and is involved in diverse biological processes including pH regulation, CO2 transfer, ionic exchange, respiration, CO2 photosynthetic fixation, and stomatal closure. Therefore, the review includes relevant aspects about CA morphology, oligomerization, and structural differences in the active site. On the other hand, we consider the general characteristics of Zn, its geometry, reactions, and physiology. We then consider the CA catalysis mechanism that is carried out by the metal ion and where Zn acts as a cofactor. Zinc deficiency can inhibit growth and protein synthesis, and there is evidence that it reduces the CA content in some plants, which is a relationship addressed in this review. In leaves, CA represents 20.1% of total soluble protein, while it is the second most abundant in the chloroplast after ribulose 1,5-disphosphate carboxylase/oxygenase (RuBisCO. This facilitates the supply of CO2 to the phosphoenolpyruvate carboxylase in C4 and CAM plants and RuBisCO in C3 plants.La anhidrasa carbónica (CA (EC: 4.2.1.1 cataliza la conversión rápida de dióxido de carbono más agua en un protón y el ion bicarbonato (HCO3-; la cual puede encontrarse en procariotas y en organismos superiores y está representada por cuatro familias distintas. La CA es una metaloenzima que requiere Zn como cofactor y está implicada en diversos procesos biológicos, incluyendo la regulación del pH, la transferencia de CO2, intercambio iónico, la respiración, la fijación fotosintética de CO2, y el cierre estomático. Por lo cual, la revisión incluye aspectos relevantes sobre la morfología de laAC, su oligomerización y diferencias estructurales en el

  12. Carbonic Anhydrases in Cnidarians: Novel Perspectives from the Octocorallian Corallium rubrum.

    Science.gov (United States)

    Le Goff, Carine; Ganot, Philippe; Zoccola, Didier; Caminiti-Segonds, Natacha; Allemand, Denis; Tambutté, Sylvie

    2016-01-01

    Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs. PMID:27513959

  13. Increased levels of carbonic anhydrase II in the developing Down syndrome brain.

    Science.gov (United States)

    Palminiello, Sonia; Kida, Elizabeth; Kaur, Kulbir; Walus, Marius; Wisniewski, Krystyna E; Wierzba-Bobrowicz, Teresa; Rabe, Ausma; Albertini, Giorgio; Golabek, Adam A

    2008-01-23

    By using a proteomic approach, we found increased levels of carbonic anhydrase II (CA II) in the brain of Ts65Dn mice, a mouse model for Down syndrome (DS). Further immunoblot analyses showed that the levels of CA II are increased not only in the brain of adult Ts65Dn mice but also in the brain of infants and young children with DS. Cellular localization of the enzyme in human brain, predominantly in the oligodendroglia and primitive vessels in fetal brain and in the oligodendroglia and some GABAergic neurons postnatally, was similar in DS subjects and controls. Given the role of CA II in regulation of electrolyte and water balance and pH homeostasis, up-regulation of CA II may reflect a compensatory mechanism mobilized in response to structural/functional abnormalities in the developing DS brain. However, this up-regulation may also have an unfavorable effect by increasing susceptibility to seizures of children with DS.

  14. Conformational effects on the circular dichroism of Human Carbonic Anhydrase II: a multilevel computational study.

    Directory of Open Access Journals (Sweden)

    Tatyana G Karabencheva-Christova

    Full Text Available Circular Dichroism (CD spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII, with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.

  15. Heavy metal ion inhibition studies of human, sheep and fish α-carbonic anhydrases.

    Science.gov (United States)

    Demirdağ, Ramazan; Yerlikaya, Emrah; Şentürk, Murat; Küfrevioğlu, Ö İrfan; Supuran, Claudiu T

    2013-04-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb(2+), Co(2+), Hg(2+), Cd(2+), Zn(2+), Se(2+), Cu(2+), Al(3+) and Mn(3+) showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate α-CAs and is probably due to their binding to His64 or the histidine cluster. PMID:22145795

  16. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  17. Carbonic anhydrase enzyme as a potential therapeutic target for experimental trichinellosis.

    Science.gov (United States)

    Saad, Abeer E; Ashour, Dalia S; Abou Rayia, Dina M; Bedeer, Asmaa E

    2016-06-01

    Trichinellosis is a globally distributed helminthic infection. There is a considerable interest in developing new anti-helminthic drugs affecting all the developmental stages of Trichinella. Acetazolamide (carbonic anhydrase (CA) inhibitor) involves a novel mechanism of action by inhibiting such an essential enzyme for parasite metabolism. This work aimed to study the effect of acetazolamide against different stages of T. spiralis in experimental animals. Mice were divided into three groups: group I: infected and treated with acetazolamide on day 2 post infection (P.I.), group II: infected and treated with acetazolamide on day 12 P.I., and group III: infected non-treated. From each group, small intestine and muscles were removed for histopathological and immunohistochemical studies. Also, total adult and muscle larval count were estimated. We found that acetazolamide was effective in reduction of both adult and muscle larval counts. When given early, the effect was more pronounced on the adults (62.7 %). However, the efficacy of the drug against muscle larvae was increased when given late (63 %). Improvement of the intestinal histopathological changes was observed in all the treated groups. Degeneration of encysted larvae with minimal pathologic changes of infected skeletal muscle was observed in the treated groups. Expression of matrix metalloproteinase-9 showed a statistically significant decrease in the intestinal and muscle tissues in all treated groups as compared to the control group. In conclusion, the present study revealed that acetazolamide, carbonic anhydrase inhibitor, could be a promising drug against both adults and larvae of T. spiralis. PMID:26979731

  18. Update and critical appraisal of combined timolol and carbonic anhydrase inhibitors and the effect on ocular blood flow in glaucoma patients

    Directory of Open Access Journals (Sweden)

    Adam M Moss

    2010-03-01

    Full Text Available Adam M Moss, Alon Harris, Brent Siesky, Deepam Rusia, Kathleen M Williamson, Yochai ShoshaniDepartment of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, USAAbstract: Topical hypotensive therapy with both timolol and carbonic anhydrase inhibitors has been shown to be efficacious at reducing intraocular pressure. Many prospective studies have also suggested that carbonic anhydrase inhibitors augment ocular blood flow and vascular regulation independent of their hypotensive effects. Although consistent in their findings, these studies must be cautiously interpreted due to the limitations of study design and specific blood flow imaging modalities. The purpose of this review is to appraise and critically evaluate the current body of literature investigating the effects of combined treatment with topical carbonic anhydrase inhibitors and timolol in patients with glaucoma with respect to ocular blood flow, visual function, and optic nerve head structure.Keywords: ocular blood flow, carbonic anhydrase inhibitor, timolol, glaucoma, visual function, optic nerve head

  19. Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase activators and polyphosphate.

    Science.gov (United States)

    Wang, Xiaohong; Schröder, Heinz C; Schlossmacher, Ute; Neufurth, Meik; Feng, Qingling; Diehl-Seifert, Bärbel; Müller, Werner E G

    2014-05-01

    Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO₃ is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO₃ formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO₃ (-)) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca²⁺ salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO₃ deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive-negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects. PMID:24374859

  20. Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

    Directory of Open Access Journals (Sweden)

    Niederhauser Barbara

    2010-07-01

    Full Text Available Abstract Background The β-carbonic anhydrase (CA, EC 4.2.1.1 enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the β-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. Results The novel β-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of β-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 × 105 s-1 and a kcat/KM of 1.1 × 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM and more potently by sulfamide (KI of 0.15 mM. Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM. Conclusions The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.

  1. The role of carbonic anhydrase in C4 photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Studer, Anthony [Life Sciences Research Foundation, Baltimore, MD (United States)

    2015-10-01

    Current pressures on the global food supply have accelerated the urgency for a second green revolution using novel and sustainable approaches to increase crop yield and efficiency. This proposal outlines experiments to address fundamental questions regarding the biology of C4 photosynthesis, the method of carbon fixation utilized by the most productive food, feed and bioenergy crops. Carbonic anhydrase (CA) has been implicated in multiple cellular functions including nitrogen metabolism, water use efficiency, and photosynthesis. CA catalyzes the first dedicated step in C4 photosynthesis, the hydration of CO2 into bicarbonate, and is potentially rate limiting in C4 grasses. Using insertional mutagenesis, we have generated CA mutants in maize, and propose the characterization of these mutants using phenotypic, physiological, and transcriptomic profiling to assay the plant’s response to altered CA activity. In addition, florescent protein tagging experiments will be employed to study the subcellular localization of CA paralogs, providing critical data for modeling carbon fixation in C4 plants. Finally, I propose parallel experiments in Setaria viridis to explore its relevance as model C4 grass. Using a multifaceted approach, this proposal addresses important questions in basic biology, as well as the need for translation research in response to looming global food challenges.

  2. The integrative segment of the quail Coturnix coturnix japonica. Occurrence and distribution of carbonic anhydrase and complex carbohydrates.

    OpenAIRE

    Gabriella, M G; Menghi, G

    1994-01-01

    As part of a more extensive study into the involvement of carbonic anhydrase in avian excretory function, the occurrence and distribution of this enzyme was investigated in the quail integrative segment. The integrative segment represents, in birds, that part of the intestinal tract where ureteral urine undergoes postrenal modification to form definitive urine. To define the structural peculiarities within the intestinal epithelium, the constituent parts, namely cloaca, rectum and caecum, as ...

  3. Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts

    OpenAIRE

    Ditte, Zuzana; Ditte, Peter; Labudova, Martina; Simko, Veronika; Iuliano, Filippo; Zatovicova, Miriam; Csaderova, Lucia; Pastorekova, Silvia; Pastorek, Jaromir

    2014-01-01

    Background Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA I...

  4. Effects of carbonyl sulfide (COS) and carbonic anhydrase on stomatal conductance

    Science.gov (United States)

    Yakir, D.; Stimler, K.; Berry, J. A.

    2011-12-01

    The potential use of COS as tracer of the gross, one-way, CO2 flux into plants is based on its co-diffusion with CO2 into leaves without outflux stimulated research on COS-CO2 interactions during leaf gas exchange. We carried out gas exchange measurements of COS and CO2 in 22 plant species representing deciduous and evergreen trees, grasses, and shrubs, under a range of light intensities and ambient COS concentrations, using mid IR laser spectroscopy. A narrow range in the normalized ratio of the net uptake rates of COS (As) and CO2 (Ac; As/Ac*[CO2]/[COS]) was observed, with a mean value of 1.61±0.26. These results reflect the dominance of stomatal conductance over both COS and CO2 uptake, imposing a relatively constant ratio between the two fluxes (except under low light conditions when CO2, but not COS, metabolism is light limited). A relatively constant ratio under common ambient conditions will facilitate the application of COS as a tracer of gross photosynthesis from leaf to global scales. However, its effect on stomatal conductance may require a special attention. Increasing COS concentrations between 250 and 2800 pmol mol-1 (enveloping atmospheric levels) seems to stimulate stomatal conductance. We examined the stimulation of conductance by COS in a range of species and show that there is a large variation with some species showing almost no response while others are highly responsive (up to doubling stomatal conductance). Using C3 and C4 plants with antisense lines abolishing carbonic anhydrase activity, we show that the activity of this enzyme is essential for both the uptake of COS and the enhancement of stomatal conductance by COS. Since carbonic anhydrase catalyzes the conversion of COS to CO2 and H2S it seems likely that the stomata are responding to H2S produced in the mesophyll. In all natural species examined the uptake of COS and CO2 were highly correlated, and there was no relationship between the sensitivity of stomata and the rate of COS uptake

  5. Anion inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Vullo, Daniela; Del Prete, Sonia; De Luca, Viviana; Carginale, Vincenzo; Ferraroni, Marta; Dedeoglu, Nurcan; Osman, Sameh M; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2016-03-01

    The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. Here we report and anion inhibition study of the β-CA, VchCAβ with anions and other small molecules which inhibit metalloenzymes. The best VchCAβ anion inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 54-86μM. Diethyldithiocarbonate was also an effective VchCAβ inhibitor, with an inhibition constant of 0.73mM. The halides, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrate, nitrite, stannate, selenate, tellurate, divanadate, tetraborate, perrhenate, perruthenate, peroxydisulfate, selenocyanide, trithiocarbonate, and fluorosulfonate showed affinity in the low millimolar range, with KIs of 2.3-9.5mM. Identification of selective inhibitors of VchCAβ (over the human CA isoforms) may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzyme. PMID:26853167

  6. Characterization of carbonic anhydrase II from Chlorella vulgaris in bio-CO2 capture.

    Science.gov (United States)

    Li, Li; Fu, Ming-Lai; Zhao, Yong-Hao; Zhu, Yun-Tian

    2012-11-01

    Carbonic anhydrase II (CA II) can catalyze the reversible hydration reaction of CO(2) at a maximum of 1.4 × 10(6) molecules of CO(2) per second. The crude intracellular enzyme extract containing CA II was derived from Chlorella vulgaris. A successful CO(2) capture experiment with the presence of calcium had been conducted on the premise that the temperature was conditioned at a scope of 30-40 °C, that the biocatalyst-nurtured algal growth period lasted 3 days, and that pH ranged from7.5 to 8.5. Ions of K(+), Na(+), Ca(2+), Co(2+), Cu(2+), Fe(3+), Mg(2+), Mn(2+), and Zn(2+) at 0.01, 0.1, and 0.5 M were found to exhibit no more than 30 % inhibition on the residual activity of the biocatalyst. It is reasonable to expect that calcification catalyzed by microalgae presents an alternative to geological carbon capture and sequestration through a chain of fundamental researches carried on under the guidance of sequestration technology. PMID:22821342

  7. Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases.

    Science.gov (United States)

    Pinard, Melissa A; Boone, Christopher D; Rife, Brittany D; Supuran, Claudiu T; McKenna, Robert

    2013-11-15

    Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I-XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity. PMID:24090602

  8. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune.

    Science.gov (United States)

    Vullo, Daniela; De Luca, Viviana; Del Prete, Sonia; Carginale, Vincenzo; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2015-04-15

    A carbonic anhydrase (CA, EC 4.2.1.1) belonging to the γ-class has been cloned, purified and characterized from the Antarctic cyanobacterium Nostoc commune. The enzyme showed a good catalytic activity for the physiologic reaction (hydration of carbon dioxide to bicarbonate and a proton) with the following kinetic parameters, kcat of 9.5×10(5)s(-1) and kcat/KM of 8.3×10(7)M(-1)s(-1), being the γ-CA with the highest catalytic activity described so far. A range of aromatic/heterocyclic sulfonamides and one sulfamate were investigated as inhibitors of the new enzyme, denominated here NcoCA. The best NcoCA inhibitors were some sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, acetazolamide, benzolamide, dorzolamide, brinzolamide and topiramate, which showed inhibition constants in the range of 40.3-92.3nM. As 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and γ-CAs are closely associated in carboxysomes of cyanobacteria for enhancing the affinity of RubisCO for CO2 and the efficiency of photosynthesis, investigation of this new enzyme and its affinity for modulators of its activity may bring new insights in these crucial processes. PMID:25773015

  9. Sclerostin regulates release of bone mineral by osteocytes by induction of carbonic anhydrase 2.

    Science.gov (United States)

    Kogawa, Masakazu; Wijenayaka, Asiri R; Ormsby, Renee T; Thomas, Gethin P; Anderson, Paul H; Bonewald, Lynda F; Findlay, David M; Atkins, Gerald J

    2013-12-01

    The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF-κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL-mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin-stimulated mineral dissolution by human primary osteocyte-like cells (hOCy) and mouse MLO-Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate-resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO-Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co-presence of the carbonic anhydrase inhibitor, acetozolamide. Car2-siRNA knockdown in MLO-Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO-Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co-addition of acetozolamide. These findings

  10. Research progress of carbon dioxide capture by using carbonic anhydrase%碳酸酐酶用于二氧化碳捕集的研究进展

    Institute of Scientific and Technical Information of China (English)

    王静

    2012-01-01

    碳酸酐酶(CA)可以加速捕集化石燃料燃烧产生的二氧化碳,从而降低CO2的排放量.主要介绍了CA的来源、活性、稳定性及作用.分析了使用新型生物方法对二氧化碳进行捕集和储存的优缺点,并对下一步的工作进行了展望.%It has been demonstrated that carbonic anhydrase has the potential of accelerating of carbon dioxide capture from fossil fuel and reduce the discharge of carbon dioxide. The source, activity, stability and functions of carbonic anhydrase are mainly presented. In addition, the advantages and disadvantages of using new biological for carbon dioxide capture and storage are discussed and analyzed, and the further study is prospected.

  11. Contribution of chloroplast biogenesis to carbon-nitrogen balance during early leaf development in rice.

    Science.gov (United States)

    Kusumi, Kensuke; Hirotsuka, Shoko; Shimada, Hiroshi; Chono, Yoko; Matsuda, Osamu; Iba, Koh

    2010-07-01

    Chloroplast biogenesis is most significant during the changes in cellular organization associated with leaf development in higher plants. To examine the physiological relationship between developing chloroplasts and host leaf cells during early leaf development, we investigated changes in the carbon and nitrogen contents in leaves at the P4 developmental stage of rice, during which leaf blade structure is established and early events of chloroplast differentiation occur. During the P4 stage, carbon content on a dry mass basis remained constant, whereas the nitrogen content decreased by 30%. Among carbohydrates, sucrose and starch accumulated to high levels early in the P4 stage, and glucose, fructose and cellulose degradation increased during the mid-to-late P4 stage. In the chloroplast-deficient leaves of the virescent-1 mutant of rice, however, the carbon and nitrogen contents, as well as the C/N ratio during the P4 stage, were largely unaffected. These observations suggest that developing rice leaves function as sink organs at the P4 stage, and that chloroplast biogenesis and carbon and nitrogen metabolism in the leaf cell is regulated independently at this stage.

  12. Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Ferraroni, Marta; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-03-01

    The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2-87.0nM. Other compounds, with medium potency against VchCAβ, (KIs in the range of 275-463nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (KIs in the range of 4.51-8.57μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes. PMID:26850377

  13. Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity.

    Science.gov (United States)

    Bozdag, Murat; Alafeefy, Ahmed M; Vullo, Daniela; Carta, Fabrizio; Dedeoglu, Nurcan; Al-Tamimi, Abdul-Malek S; Al-Jaber, Nabila A; Scozzafava, Andrea; Supuran, Claudiu T

    2015-12-15

    Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with KIs of 7.6-322nM against hCA I, of 0.06-85.4nM against hCA II; of 6.7-152nM against hCA IX and of 0.49-237nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail. PMID:26639945

  14. Synthesis and Evaluation of New Phthalazine Urea and Thiourea Derivatives as Carbonic Anhydrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Nurcan Berber

    2013-01-01

    Full Text Available A new series of phthalazine substituted urea and thiourea derivatives were synthesized, and their inhibitory effects on the activity of purified human carbonic anhydrases (hCAs I and II were evaluated. 2H-Indazolo[2,1-b]phthalazine-trione derivative (1 was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and nitro group was reduced to amine derivative (2 with SnCl2·2H2O. The compound was reacted with isocyanates and isothiocyanates to get the final products (3a–p. The results showed that all the synthesized compounds inhibited the CA isoenzymes activity. 3a (IC50 = 6.40 µM for hCA I and 6.13 µM for hCA II has the most inhibitory effect. The synthesized compounds are very bulky to be able to bind near the zinc ion, and they much more probably bind as the coumarin derivatives.

  15. Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II.

    Science.gov (United States)

    Taraphder, Srabani; Maupin, C Mark; Swanson, Jessica M J; Voth, Gregory A

    2016-08-25

    The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II. PMID:27063577

  16. Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

    Directory of Open Access Journals (Sweden)

    Tomas Eija

    2004-10-01

    Full Text Available Abstract Background Carbonic anhydrase (CA classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

  17. Enzyme renaturation to higher activity driven by the sol-gel transition: Carbonic anhydrase

    Science.gov (United States)

    Vinogradov, Vladimir V.; Avnir, David

    2015-09-01

    We describe a so-far unknown route for renaturing denatured enzymes, namely subjecting the denatured enzyme to an oxide sol-gel transition. The phenomenon was revealed in a detailed study of denatured carbonic anhydrase which was subjected to an alumina sol-gel transition, up to the thermally stabilizing entrapment in the final xerogel. Remarkably, not only that the killed enzyme regained its activity during the sol-gel process, but its activity increased to 180% of the native enzyme. To the best of our knowledge, this is the first report of enhanced activity following by renaturing (a “Phoenix effect”). Kinetic study which revealed a five-orders of magnitude (!) increase in the Arrhenius prefactor upon entrapment compared to solution. Circular dichroism analysis, differential scanning calorimetry, zeta potential analyses as well as synchronous fluorescence measurements, all of which were used to characterize the phenomenon, are consistent with a proposed mechanism which is based on the specific orienting interactions of the active site of the enzyme with respect to the alumina interface and its pores network.

  18. Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase.

    Science.gov (United States)

    Bozdag, Murat; Carta, Fabrizio; Vullo, Daniela; Isik, Semra; AlOthman, Zeid; Osman, Sameh M; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-01

    Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, were investigated for the inhibition of α- and β-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the Saccharomyces cerevisiae β-CA, scCA. The yeast and its β-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA I inhibitors (K(I)s of 66.5-910 nM), were more effective as hCA II inhibitors (K(I)s of 8.9-107 nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, with inhibition constants ranging between 6.4 and 259 nM. The detailed structure activity relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms. PMID:25669351

  19. Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Ferraroni, Marta; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-03-01

    The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2-87.0nM. Other compounds, with medium potency against VchCAβ, (KIs in the range of 275-463nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (KIs in the range of 4.51-8.57μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes.

  20. Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    Science.gov (United States)

    Vullo, Daniela; De Luca, Viviana; Del Prete, Sonia; Carginale, Vincenzo; Scozzafava, Andrea; Osman, Sameh M; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2016-02-15

    The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(6) M(-1) s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K(I) of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here, by allowing the identification of inhibitors which may be useful as pharmacologic tools.

  1. Comparison of amino and epoxy functionalized SBA-15 used for carbonic anhydrase immobilization.

    Science.gov (United States)

    Fei, Xiaoyao; Chen, Shaoyun; Liu, Dai; Huang, Chunjie; Zhang, Yongchun

    2016-09-01

    Two functionalized SBA-15 [amine-functionalized SBA-15 (AFS) and epoxy-functionalized SBA-15 (GFS)] with different types of functional groups were synthesized by a hydrothermal process and post functionalized with 3-aminopropyltriethoxysilane (APTES) and 3-glycidyloxypropyltrimethoxysilane (GPTMS), respectively. They were used for the immobilization of carbonic anhydrase (CA). The physicochemical properties of the functionalized SBA-15 were characterized by X-ray powder diffraction (XRD), N2 adsorption-desorption, (13)C, (29)Si solid-state nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). Before and after CA was immobilized on AFS and GFS, the effects of temperature and pH value on the enzyme activity, storage stability, and reusability were investigated using para-nitrophenyl acetate (p-NPA) assay. CA/GFS showed a better performance with respect to storage stability and reusability than CA/AFS. Moreover, the amount of CaCO3 precipitated over CA/AFS was less than that precipitated over CA/GFS, which was almost equal to that precipitated over the free CA. The results indicate that the epoxy group is a more suitable functional group for covalent bonding with CA than the amino group, and GFS is a promising support for CA immobilization. PMID:27215831

  2. Spectroscopic and MD simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA induced by urea.

    Science.gov (United States)

    Idrees, Danish; Prakash, Amresh; Haque, Md Anzarul; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

    2016-09-01

    Carbonic anhydrase VA (CAVA) is primarily expressed in the mitochondria and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis and neuronal transmission. To understand the biophysical properties of CAVA, we carried out a reversible urea-induced isothermal denaturation at pH 7.0 and 25°C. Spectroscopic probes, [θ]222 (mean residue ellipticity at 222 nm), F344 (Trp-fluorescence emission intensity at 344 nm) and Δε280 (difference absorption at 280 nm) were used to monitor the effect of urea on the structure and stability of CAVA. The urea-induced reversible denaturation curves were used to estimate [Formula: see text], Gibbs free energy in the absence of urea; Cm, the mid-point of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Coincidence of normalized transition curves of all optical properties suggests that unfolding/refolding of CAVA is a two-state process. We further performed 40 ns molecular dynamics simulation of CAVA to see the dynamics at different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.

  3. Innovative molecular diagnosis of Trichinella species based on β-carbonic anhydrase genomic sequence.

    Science.gov (United States)

    Zolfaghari Emameh, Reza; Kuuslahti, Marianne; Näreaho, Anu; Sukura, Antti; Parkkila, Seppo

    2016-03-01

    Trichinellosis is a helminthic infection where different species of Trichinella nematodes are the causative agents. Several molecular assays have been designed to aid diagnostics of trichinellosis. These assays are mostly complex and expensive. The genomes of Trichinella species contain certain parasite-specific genes, which can be detected by polymerase chain reaction (PCR) methods. We selected β-carbonic anhydrase (β-CA) gene as a target, because it is present in many parasites genomes but absent in vertebrates. We developed a novel β-CA gene-based method for detection of Trichinella larvae in biological samples. We first identified a β-CA protein sequence from Trichinella spiralis by bioinformatic tools using β-CAs from Caenorhabditis elegans and Drosophila melanogaster. Thereafter, 16 sets of designed primers were tested to detect β-CA genomic sequences from three species of Trichinella, including T. spiralis, Trichinella pseudospiralis and Trichinella nativa. Among all 16 sets of designed primers, the primer set No. 2 efficiently amplified β-CA genomic sequences from T. spiralis, T. pseudospiralis and T. nativa without any false-positive amplicons from other parasite samples including Toxoplasma gondii, Toxocara cati and Parascaris equorum. This robust and straightforward method could be useful for meat inspection in slaughterhouses, quality control by food authorities and medical laboratories. PMID:26639312

  4. Indazole, Pyrazole, and Oxazole Derivatives Targeting Nitric Oxide Synthases and Carbonic Anhydrases.

    Science.gov (United States)

    Maccallini, Cristina; Di Matteo, Mauro; Vullo, Daniela; Ammazzalorso, Alessandra; Carradori, Simone; De Filippis, Barbara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Supuran, Claudiu T; Amoroso, Rosa

    2016-08-19

    Nitric oxide (NO) is an essential endogenous mediator with a physiological role in the central nervous system as neurotransmitter and neuromodulator. A growing number of studies have demonstrated that abnormal nitrergic signaling is a crucial event in the development of neurodegeneration. In particular, the uncontrolled production of NO by neuronal nitric oxide synthase (nNOS) is observed in several neurodegenerative diseases. Moreover, it is well recognized that specific isoforms of human carbonic anhydrase (hCA) physiologically modulate crucial pathways of signal processing and that low expression of CA affects cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive impairments. In light of this, dual agents that are able to target both NOS (inhibition) and CA (activation) could be useful drug candidates for the treatment of Alzheimer's disease, aging, and other neurodegenerative diseases. In the present work, we show the design, synthesis, and in vitro biological evaluation of new nitrogen-based heterocyclic compounds. Among the tested molecules, 2-amino-3-(4-hydroxyphenyl)-N-(1H-indazol-5-yl)propanamide hydrochloride (10 b) was revealed to be a potent dual agent, able to act as a selective nNOS inhibitor and activator of the hCA I isoform. PMID:27377568

  5. Sulfamate inhibitor S4 influences carbonic anhydrase IX ectodomain shedding in colorectal carcinoma cells.

    Science.gov (United States)

    Hektoen, Helga Helseth; Ree, Anne Hansen; Redalen, Kathrine Røe; Flatmark, Kjersti

    2016-10-01

    Carbonic anhydrase IX (CAIX) is a pivotal pH regulator under hypoxia, which by its tumor-specific expression represents an attractive target for cancer therapy. Here, we report on effects of the sulfamate CAIX inhibitor S4 (4-(3'-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate) in colorectal carcinoma cell lines. S4 was administered under experimental hypoxia or normoxia to HT29, KM20L2 and HCT116 cells. Effects on survival, proliferation, pH, lactate extrusion and CAIX protein expression were evaluated. S4 treatment resulted in attenuated hypoxia-induced extracellular acidification and reduced clonogenic survival under hypoxia in HT29 cells. The pH effects were present only in a [Formula: see text]-free buffer system and were accompanied by decreased lactate extrusion. The main finding of this work was that S4 treatment caused alterations in CAIX ectodomain shedding. This merits further investigation to understand how sulfamates influence CAIX activity and how such drugs may be of use in cancer treatment. PMID:26244271

  6. Coumarin or benzoxazinone based novel carbonic anhydrase inhibitors: synthesis, molecular docking and anticonvulsant studies.

    Science.gov (United States)

    Karataş, Mert Olgun; Uslu, Harun; Sarı, Suat; Alagöz, Mehmet Abdullah; Karakurt, Arzu; Alıcı, Bülent; Bilen, Cigdem; Yavuz, Emre; Gencer, Nahit; Arslan, Oktay

    2016-10-01

    Among many others, coumarin derivatives are known to show human carbonic anhydrase (hCA) inhibitory activity. Since hCA inhibition is one of the underlying mechanisms that account for the activities of some antiepileptic drugs (AEDs), hCA inhibitors are expected to have anti-seizure properties. There are also several studies reporting compounds with an imidazole and/or benzimidazole moiety which exert these pharmacological properties. In this study, we prepared fifteen novel coumarin-bearing imidazolium and benzimidazolium chloride, nine novel benzoxazinone-bearing imidazolium and benzimidazolium chloride derivatives and evaluated their hCA inhibitory activities and along with fourteen previously synthesized derivatives we scanned their anticonvulsant effects. As all compounds inhibited purified hCA isoforms I and II, some of them also proved protective against Maximal electroshock seizure (MES) and ScMet induced seizures in mice. Molecular docking studies with selected coumarin derivatives have revealed that these compounds bind to the active pocket of the enzyme in a similar fashion to that previously described for coumarin derivatives.

  7. Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: novel sulfamoylcarbamates and sulfamides derived from acetophenones.

    Science.gov (United States)

    Akıncıoğlu, Akın; Akıncıoğlu, Hülya; Gülçin, İlhami; Durdagi, Serdar; Supuran, Claudiu T; Göksu, Süleyman

    2015-07-01

    In this study, several novel sulfamides were synthesized and evaluated for their acetylcholine esterase (AChE) and human carbonic anhydrase I, and II isoenzymes (hCA I and II) inhibition profiles. Reductive amination of methoxyacetophenones was used for the synthesis of amines. Amines were converted to sulfamoylcarbamates with chlorosulfonyl isocyanate (CSI) in the presence of BnOH. Pd-C catalyzed hydrogenolysis of sulfamoylcarbamates afforded sulfamides. These novel compounds were good inhibitors of the cytosolic hCA I, and hCA II with Ki values in the range of 45.9±8.9-687.5±84.3 pM for hCA I, and 48.80±8.2-672.2±71.9pM for hCA II. The inhibitory effects of the synthesized novel compounds on AChE were also investigated. The Ki values of these compounds were in the range of 4.52±0.61-38.28±6.84pM for AChE. These results show that hCA I, II, and AChE were effectively inhibited by the novel sulfamoylcarbamates 17-21 and sulfamide derivatives 22-26. All investigated compounds were docked within the active sites of the corresponding enzymes revealing the reasons of the effective inhibitory activity. PMID:25921269

  8. Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa

    International Nuclear Information System (INIS)

    Carbonic anhydrase (CA) isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated

  9. Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa

    Directory of Open Access Journals (Sweden)

    Saarnio Juha

    2005-04-01

    Full Text Available Abstract Background Carbonic anhydrase (CA isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. Methods Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. Results The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. Conclusion The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated.

  10. Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii.

    Science.gov (United States)

    Vullo, Daniela; Del Prete, Sonia; Osman, Sameh M; De Luca, Viviana; Scozzafava, Andrea; Alothman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2014-01-01

    The δ-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a δ-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with KIs of 49.6-118nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (KIs of 375-897nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with KIs of 4.27-9.20μM. The inhibition profile of the δ-CA is very different from that of α-, β- and γ-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the δ-CA active site. PMID:24314394

  11. Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum.

    Science.gov (United States)

    Vullo, Daniela; Del Prete, Sonia; Fisher, Gillian M; Andrews, Katherine T; Poulsen, Sally-Ann; Capasso, Clemente; Supuran, Claudiu T

    2015-02-01

    The η-carbonic anhydrases (CAs, EC 4.2.1.1) were recently discovered as the sixth genetic class of this metalloenzyme superfamily, and are so far known only in protozoa, including various Plasmodium species, the causative agents of malaria. We report here an inhibition study of the η-CA from Plasmodium falciparum (PfCA) against a panel of sulfonamides and one sulfamate compound, some of which are clinically used. The strongest inhibitors identified were ethoxzolamide and sulthiame, with KIs of 131-132 nM, followed by acetazolamide, methazolamide and hydrochlorothiazide (KIs of 153-198 nM). Brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM. An interesting observation was that the more efficient PfCA inhibitors are representative of several scaffolds and chemical classes, including benzene sulfonamides, monocyclic/bicyclic heterocyclic sulfonamides and compounds with a more complex scaffold (i.e., the sugar sulfamate derivative, topiramate, and the coxibs, celecoxib and valdecoxib). A comprehensive inhibition study of small molecules for η-CAs is needed as a first step towards assessing PfCA as a druggable target. The present work identifies the first known η-CA inhibitors and provides a platform for the development of next generation novel PfCA inhibitors. PMID:25533402

  12. Influence of pesticide exposure on carbonic anhydrase II from sheep stomach.

    Science.gov (United States)

    Kılınç, Namık; İşgör, Mehmet Mustafa; Şengül, Bülent; Beydemir, Şükrü

    2015-09-01

    Carbonic anhydrase (CA) is a widely distributed enzyme and has a crucial role in the cells, tissues and organs of living organisms. It is found that CA-II is one of the most abundant CA isoenzymes in the gastrointestinal system. It plays an important role in the gastric acid secretion in stomach. In this study, we purified CA-II isoenzyme from sheep stomach with a 615.2 purification fold, 78% purification yield and 5562.02 specific activity. Moreover, the in vitro effects of some commonly used pesticides including chlorpyrifos, cypermethrin, dichlorvos, glyphosate isopropylamine and lambda cyhalomethrin on the enzyme activity were investigated. Of these compounds, glyphosate isopropylamine and dichlorvos showed an inhibition on CA-II esterase activity. They have IC50 values of 0.155 µM and 2.690 µM and Ki values of 0.329 µM and 3.654 µM, respectively. Both glyphosate isopropylamine and dichlorvos inhibited CA-II isoenzyme in a noncompetitive manner.

  13. Carbonic anhydrase XII expression is associated with histologic grade of cervical cancer and superior radiotherapy outcome

    International Nuclear Information System (INIS)

    To investigate whether expression of carbonic anhydrase XII (CA12) is associated with histologic grade of the tumors and radiotherapy outcomes of the patients with invasive cervical cancer. CA12 expression was examined by immunohistochemical stains in cervical cancer tissues from 183 radiotherapy patients. Histological grading was classified as well (WD), moderately (MD) or poorly differentiated (PD). Oligonucleotide microarray experiment was performed using seven cervical cancer samples to examine differentially expressed genes between WD and PD cervical cancers. The association between CA12 and histological grade was analyzed by chi-square test. CA12 and histological grades were analyzed individually and as combined CA12 and histologic grade categories for effects on survival outcome. Immunohistochemical expression of CA12 was highly associated with the histologic grade of cervical cancer. Lack of CA12 expression was associated with PD histology, with an odds ratio of 3.9 (P = 0.01). Microarray analysis showed a fourfold reduction in CA12 gene expression in PD tumors. CA12 expression was marginally associated with superior disease-free survival. Application of the new combined categories resulted in further discrimination of the prognosis of patients with moderate and poorly differentiated tumor grade. Our study indicates that CA12 may be used as a novel prognostic marker in combination with histologic grade of the tumors

  14. Membrane Specific Carbonic Anhydrase (CA-IV) Expression in Bovine Lung: The Effects of Alcoholic and Non-Alcoholic Drinks

    OpenAIRE

    DEMİR, Nazan; NADAROĞLU, Hayrunnisa

    2002-01-01

    Carbonic anhydrase (CA) (carbonate hydrolyase: E. C. 4.2.1.1) from bovine lung was purified by a new method and characterized. The purification level was 4306-fold. The optimum temperature for maximum enzyme activity was 37.5°C. The optimum pH was 7.4, varying between 3.5 and 10.0. SDS-polyacryamide gel electrophoresis (3-10% discontinuous SDS-PAGE) showed two distinct bands for CA-IV. The molecular weights of the enzymes were found to be approximately 54.000 and 29.000, respectively. ...

  15. Role of Carbonic Anhydrase as an Activator in Carbonate Rock Dissolution and Its Implication for Atmospheric CO2 Sink

    Institute of Scientific and Technical Information of China (English)

    刘再华

    2001-01-01

    The conversion of CO2 into H+ and is a relatively slow reaction. Hence, its kinetics may be rate determining in carbonate rock dissolution. Carbonic anhydrase (CA), which is widespread in nature, was used to catalyze the CO2 conversion process in dissolution experiments of limestone and dolomite. It was found that the rate of dissolution increases by a factor of about 10 after the addition of CA at a high CO2 partial pressure (Pco2) for limestone and about 3 at low Pco2 for dolomite. This shows that reappraisal is necessary for the importance of chemical weathering (including carbonate rock dissolution and silicate weathering) in the atmospheric CO2 sink and the mysterious missing sink in carbon cycling. It is doubtless that previous studies of weathering underestimated weathering rates due to the ignorance of CA as an activator in weathering, thus the contribution of weathering to the atmospheric CO2 sink is also underestimated. This finding also shows the need to examine the situ distribution and activity of CA in different waters and to investigate the role of CA in weathering.``

  16. Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

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    Amaya Blanco-Rivero

    Full Text Available BACKGROUND: Cah3 is the only carbonic anhydrase (CA isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2 conditions. RESULTS/CONCLUSIONS: In the present work we demonstrate that after transfer to low CO(2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2 conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. SIGNIFICANCE: This is the first report of a CA being post

  17. Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos.

    Science.gov (United States)

    Karakostis, Konstantinos; Costa, Caterina; Zito, Francesca; Brümmer, Franz; Matranga, Valeria

    2016-06-01

    Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus. PMID:27230618

  18. Enzyme-accelerated and structure-guided crystallization of calcium carbonate: role of the carbonic anhydrase in the homologous system.

    Science.gov (United States)

    Müller, Werner E G; Schlossmacher, Ute; Schröder, Heinz C; Lieberwirth, Ingo; Glasser, Gunnar; Korzhev, Michael; Neufurth, Meik; Wang, Xiaohong

    2014-01-01

    The calcareous spicules from sponges, e.g. from Sycon raphanus, are composed of almost pure calcium carbonate. In order to elucidate the formation of those structural skeletal elements, the function of the enzyme carbonic anhydrase (CA), isolated from this species, during the in vitro calcium carbonate-based spicule formation, was investigated. It is shown that the recombinant sponge CA substantially accelerates calcium carbonate formation in the in vitro diffusion assay. A stoichiometric calculation revealed that the turnover rate of the sponge CA during the calcification process amounts to 25 CO2s(-1) × molecule CA(-1). During this enzymatically driven process, initially pat-like particles are formed that are subsequently transformed to rhomboid/rhombohedroid crystals with a dimension of ~50 μm. The CA-catalyzed particles are smaller than those which are formed in the absence of the enzyme. The Martens hardness of the particles formed is ~4 GPa, a value which had been determined for other biogenic calcites. This conclusion is corroborated by energy-dispersive X-ray spectroscopy, which revealed that the particles synthesized are composed predominantly of the elements calcium, oxygen and carbon. Surprising was the finding, obtained by light and scanning electron microscopy, that the newly formed calcitic crystals associate with the calcareous spicules from S. raphanus in a highly ordered manner; the calcitic crystals almost perfectly arrange in an array orientation along the two opposing planes of the spicules, leaving the other two plane arrays uncovered. It is concluded that the CA is a key enzyme controlling the calcium carbonate biomineralization process, which directs the newly formed particles to existing calcareous spicular structures. It is expected that with the given tools new bioinspired materials can be fabricated. PMID:23978410

  19. Immunocytochemical localization of carbonic anhydrase in the pseudobranch tissue of the rainbow trout Oncorhynchus mykiss

    Institute of Scientific and Technical Information of China (English)

    S. M. RAHIM; A. G. MAZLAN; K. D. SIMON; J. P. DELAUNOY; P. LAURENT

    2014-01-01

    本文题目:虹鳟假鳃组织中的碳酸酐酶免疫细胞化学定位Immunocytochemical localization of carbonic anhydrase in the pseudobranch tissue of the rainbow trout Oncorhynchus mykiss研究目的:假腮的功能早已引起科学家兴趣,但还有待阐明。本文通过研究硬骨鱼类品种虹鳟鱼(Oncorhynchus mykiss)的假腮碳酸酐酶的免疫定位,来探讨假腮碳酸酐酶的生理功能。研究方法:免疫组织化学染色技术。重要结论:免疫组化结果显示碳酸酐酶分布在假腮细胞中,更精确地说是在其细胞顶端分布。细胞基底端、管状系统、毛细血管和柱细胞均无免疫染色。免疫细胞化学定位进一步验证了这些结果,并显示一部分是细胞质碳酸酐酶,其余的与细胞膜结构连接。此外,腔隙层未显示出免疫过氧化物酶的活性。本研究揭示了假腮碳酸酐酶的功能与细胞外介质有关,碳酸酐酶能干预传入神经纤维刺激机制。%Pseudobranch function has long interested scientists, but its role has yet to be elucidated. Several studies have suggested that pseudobranchs serve respiratory, osmoregulatory, and sensory functions. This work investigated the immunolocalization of pseudobranch carbonic anhydrase (CA) in the teleost fish species rainbow trout (Oncor-hynchus mykiss) to clarify its physiological function. CA was purified from rainbow trout gil s O. mykiss and specific antibodies were raised. Immunoblotting between tissue homogenates of pseudobranch and gil CA antibodies showed specific immunostaining with only one band corresponding to CA in the pseudobranch homogenate. Results of im-munohistochemical technique revealed that CA was distributed within pseudobranch cells and more precisely in the apical parts (anti-vascular) of cells. The basal (vascular) parts of cells, tubular system, blood capillaries, and pillar cells were not immunostained. Immunocytochemistry confirmed these results and

  20. Carbonic anhydrase II deficiency: Single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients

    Energy Technology Data Exchange (ETDEWEB)

    Hu, P.Y.; Ernst, A.R.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (United States)); Venta, P.J. (Michigan State Univ., East Lansing, MI (United States)); Skaggs, L.A.; Tashian, R.E. (Univ. of Michigan Medical School, Ann Arbor, MI (United States))

    1994-04-01

    To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. The authors report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation. These findings identify a novel mutation common to Hispanic patients from the Caribbean islands and provide a ready means for PCR-based diagnosis of the [open quotes]Hispanic mutation.[close quotes] The basis for their phenotypic variability is not yet clear. 15 refs., 5 figs., 1 tab.

  1. Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-09-15

    We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO2 hydration: PfCAdom showed a kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), whereas PfCA showed a lower activity compared to PfCAdom, with a kcat of 1.4×10(5)s(-1) and kcat/Km of 5.4×10(6)M(-1)×s(-1). PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 9-68μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with KIs in the range of 0.53-0.97mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action. PMID:27480028

  2. Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

    Directory of Open Access Journals (Sweden)

    Claes-Göran Reibring

    Full Text Available Carbonic anhydrases (CAs play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for

  3. Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

    Directory of Open Access Journals (Sweden)

    Mayank Aggarwal

    2014-03-01

    Full Text Available Human carbonic anhydrases (CAs are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type of a variant of CA II in which His64 is replaced with Ala (H64A CA II can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations the activity of H64A CA II.

  4. Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

    Science.gov (United States)

    Esbaugh, A J; Secor, S M; Grosell, M

    2015-09-01

    Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy.

  5. Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis

    OpenAIRE

    Yu, Su-jong; Yoon, Jung-Hwan; Lee, Jeong-Hoon; Myung, Sun-jung; Jang, Eun-sun; Kwak, Min-Sun; Cho, Eun-Ju; Jang, Ja-June; Kim, Yoon-jun; Lee, Hyo-Suk

    2011-01-01

    Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth wa...

  6. Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma.

    Science.gov (United States)

    Vullo, Daniela; Durante, Mariaconcetta; Di Leva, Francesco Saverio; Cosconati, Sandro; Masini, Emanuela; Scozzafava, Andrea; Novellino, Ettore; Supuran, Claudiu T; Carta, Fabrizio

    2016-06-23

    A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines and COS as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates, the xanthates, and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the pharmacologically relevant human (h) hCAs isoforms I, II, IX, and XII in vitro and showed KIs spanning between the low and medium nanomolar range. By means of a computational study, the MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering effects and showed interesting activity. PMID:27253845

  7. Linking Carbonic Anhydrase Abundance and Diversity in Soils to Ecological Function

    Science.gov (United States)

    Pang, E.; Meredith, L. K.; Welander, P. V.

    2015-12-01

    Carbonic anhydrase (CA) is an ancient enzyme widespread among bacteria, archaea, and eukarya that catalyzes the following reaction: CO2 + H2O ⇌ HCO3- + H+. Its functions are critical for key cellular processes such as concentrating CO2 for autotrophic growth, pH regulation, and pathogen survival in hosts. Currently, there are six known CA classes (α, β, γ, δ, η, ζ) arising from several distinct evolutionary lineages. CA are widespread in sequenced genomes, with many organisms containing multiple classes of CA or multiple CA of the same class. Soils host rich microbial communities with diverse and important ecological functions, but the diversity and abundance of CA in soils has not been explored. CA appears to play an important, but poorly understood, role in some biogeochemical cycles such as those of CO2 and its oxygen isotope composition and also carbonyl sulfide (COS), which are potential tracers in predictive carbon cycle models. Recognizing the prevalence and functional significance of CA in soils, we used a combined bioinformatics and molecular biology approach to address fundamental questions regarding the abundance, diversity, and function of CA in soils. To characterize the abundance and diversity of the different CA classes in soils, we analyzed existing soil metagenomic and metatranscriptomic data from the DOE Joint Genome Institute databases. Out of the six classes of CA, we only found the α, β, and γ classes to be present in soils, with the β class being the most abundant. We also looked at genomes of sequenced soil microorganisms to learn what combination of CA classes they contain, from which we can begin to predict the physiological role of CA. To characterize the functional roles of the different CA classes in soils, we collected soil samples from a variety of biomes with diverse chemical and physical properties and quantified the rate of two CA-mediated processes: soil uptake of COS and acceleration of the oxygen isotope exchange

  8. Surface Engineering of Polypropylene Membranes with Carbonic Anhydrase-Loaded Mesoporous Silica Nanoparticles for Improved Carbon Dioxide Hydration.

    Science.gov (United States)

    Yong, Joel K J; Cui, Jiwei; Cho, Kwun Lun; Stevens, Geoff W; Caruso, Frank; Kentish, Sandra E

    2015-06-01

    Carbonic anhydrase (CA) is a native enzyme that facilitates the hydration of carbon dioxide into bicarbonate ions. This study reports the fabrication of thin films of active CA enzyme onto a porous membrane substrate using layer-by-layer (LbL) assembly. Deposition of multilayer films consisting of polyelectrolytes and CA was monitored by quartz crystal microgravimetry, while the enzymatic activity was assayed according to the rates of p-nitrophenylacetate (p-NPA) hydrolysis and CO2 hydration. The fabrication of the films onto a nonporous glass substrate showed CO2 hydration rates of 0.52 ± 0.09 μmol cm(-2) min(-1) per layer of bovine CA and 2.6 ± 0.7 μmol cm(-2) min(-1) per layer of a thermostable microbial CA. The fabrication of a multilayer film containing the microbial CA on a porous polypropylene membrane increased the hydration rate to 5.3 ± 0.8 μmol cm(-2) min(-1) per layer of microbial CA. The addition of mesoporous silica nanoparticles as a film layer prior to enzyme adsorption was found to increase the activity on the polypropylene membranes even further to a rate of 19 ± 4 μmol cm(-2) min(-1) per layer of microbial CA. The LbL treatment of these membranes increased the mass transfer resistance of the membrane but decreased the likelihood of membrane pore wetting. These results have potential application in the absorption of carbon dioxide from combustion flue gases into aqueous solvents using gas-liquid membrane contactors. PMID:25984966

  9. Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker.

    Science.gov (United States)

    Azevedo-Linhares, Maristela; Freire, Carolina A

    2015-12-01

    In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present.

  10. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Christopher D. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); Tu, Chingkuang [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)

    2014-06-01

    The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.

  11. Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

    Directory of Open Access Journals (Sweden)

    Lerman Michael I

    2009-03-01

    Full Text Available Abstract Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1. Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1 HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2 There is no evidence of

  12. Downregulation of carbonic anhydrase IX promotes Col10a1 expression in chondrocytes.

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    Toshifumi Maruyama

    Full Text Available Carbonic anhydrase (CA IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO(2. While it is known that CA IX is distributed in human embryonic chondrocytes, its role in chondrocyte differentiation has not been reported. In the present study, we found that Car9 mRNA and CA IX were expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation in vitro. Introduction of Car9 siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of Col10a1, the gene for type X collagen α-1 chain, and Epas1, the gene for hypoxia-responsible factor-2α (HIF-2α, both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of Col10a1 and Epas1, while the transcription of Col2a1 and Acan were up-regulated. Although HIF-2α has been reported to be a potent activator of Col10a1 transcription, Epas1 siRNA did not suppress Car9 siRNA-induced increment in Col10a1 expression, indicating that down-regulation of CA IX induces the expression of Col10a1 in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by Car9 siRNA. Furthermore, the expression of Col10a1 mRNA after Car9 silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of Col10a1 expression by down-regulation of Car9, more detailed study is required to clarify the role of CA IX in regulation of Col10a1 expression in chondrocytes.

  13. Ethylene bis-imidazoles are highly potent and selective activators for isozymes VA and VII of carbonic anhydrase, with a potential nootropic effect

    OpenAIRE

    Draghici, Bogdan; Vullo, Daniela; Akocak, Suleyman; Walker, Ellen A; Supuran, Claudiu T.; Ilies, Marc A.

    2014-01-01

    A series of ethylene bis-imidazoles was synthesized via a novel microwave-mediated synthesis. Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against CA VA and CA VII, also displaying excellent selectivity against other CA isozymes present in this organ.

  14. Influence of Carbonic Anhydrase Activity in Terrestrial Vegetation on the 18O Content of Atmospheric CO2

    Science.gov (United States)

    Gillon, Jim; Yakir, Dan

    2001-03-01

    The oxygen-18 (18O) content of atmospheric carbon dioxide (CO2) is an important indicator of CO2 uptake on land. It has generally been assumed that during photosynthesis, oxygen in CO2 reaches isotopic equilibrium with oxygen in 18O-enriched water in leaves. We show, however, large differences in the activity of carbonic anhydrase (which catalyzes CO2 hydration and 18O exchange in leaves) among major plant groups that cause variations in the extent of 18O equilibrium (θeq). A clear distinction in θeq between C3 trees and shrubs, and C4 grasses makes atmospheric C18OO a potentially sensitive indicator to changes in C3 and C4 productivity. We estimate a global mean θeq value of ~0.8, which reasonably reconciles inconsistencies between 18O budgets of atmospheric O2 (Dole effect) and CO2.

  15. Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase.

    Science.gov (United States)

    Guo, C; Gynn, M; Chang, T M S

    2015-06-01

    We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb-SOD-CAT-CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol-chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb-SOD-CAT-CA to 2, 4, or 6 times that of RBC.

  16. Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes

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    Orito Kensuke

    2011-03-01

    Full Text Available Abstract Background Carbonic anhydrase (CA of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes. Methods Chicken CA-II was purified by a combination of column chromatography. The levels of CA-II in the hemolysate of the chicken were determined using the ELISA system in blood samples from 279 female chickens, ages 1 to 93 weeks, 69 male chickens, ages 3 to 59 weeks and 52 weeks female Araucana-chickens. Results The mean concentration of CA-II in hemolysate from 1-week-old female was 50.8 ± 11.9 mg/g of Hb. The mean levels of CA-II in 25-week-old (188.1 ± 82.6 mg/g of Hb, 31-week-old (193.6 ± 69.7 mg/g of Hb and 49-week-old (203.8 ± 123.5 mg/g of Hb female-chickens showed the highest level of CA-II. The levels of CA-II in female WL-chickens significantly decreased at 63 week (139.0 ± 19.3 mg/g of Hb. The levels of CA-II in female WL-chicken did not change from week 63 until week 93.The mean level of CA-II in hemolysate of 3-week-old male WL-chickens was 78.3 ± 20.7 mg/g of Hb. The levels of CA-II in male WL-chickens did not show changes in the week 3 to week 59 timeframe. The mean level of CA-II in 53-week-old female Araucana-chickens was 23.4 ± 1.78 mg/g of Hb. These levels of CA-II were about 11% of those of 49-week-old female WL-chickens. Simple linear regression analysis showed significant associations between the level of CA-II and egg laying rate from 16 week-old at 63 week-old WL-chicken (p Conclusions Developmental changes and sexual differences of CA-II concentration in WL-chicken erythrocytes were observed. The concentration of CA-II in

  17. Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors.

    Science.gov (United States)

    Ceruso, Mariangela; Antel, Sabrina; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-01

    New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with KIs in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents. PMID:25792500

  18. Size and surface chemistry of nanoparticles lead to a variant behavior in the unfolding dynamics of human carbonic anhydrase

    Science.gov (United States)

    Nasir, Irem; Lundqvist, Martin; Cabaleiro-Lago, Celia

    2015-10-01

    The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence revealed that HCAs adsorb to both hydrophilic and hydrophobic surfaces, however the dynamics of the unfolding at the nanoparticle surfaces drastically vary with the polarity. The size of the nanoparticles has opposite effects depending on the polarity of the nanoparticle surface. The apolar nanoparticles induce seconds timescale structural rearrangements whereas polar nanoparticles induce hours timescale structural rearrangements on the same charged HCA variant. Here, a simple model is proposed where the difference in the timescales of adsorption is correlated with the energy barriers for initial docking and structural rearrangements which are firmly regulated by the surface polarity. Near-UV circular dichorism (CD) further supports that both protein variants undergo structural rearrangements at the nanoparticle surfaces regardless of being ``hard'' or ``soft''. However, the conformational changes induced by the apolar surfaces differ for each HCA isoform and diverge from the previously reported effect of silica nanoparticles.The adsorption induced conformational changes of human carbonic anhydrase I (HCAi) and pseudo wild type human carbonic anhydrase II truncated at the 17th residue at the N-terminus (trHCAii) were studied in presence of nanoparticles of different sizes and polarities. Isothermal titration calorimetry (ITC) studies showed that the binding to apolar surfaces is affected by the nanoparticle size in combination with the inherent protein stability. 8-Anilino

  19. Novel sulfonamide bearing coumarin scaffolds as selective inhibitors of tumor associated carbonic anhydrase isoforms IX and XII.

    Science.gov (United States)

    Chandak, Navneet; Ceruso, Mariangela; Supuran, Claudiu T; Sharma, Pawan K

    2016-07-01

    Four novel scaffolds consisting of total 24 compounds (1a-1o, 2a-2c, 3a-3c and 4a-4c) bearing aromatic sulfonamide and coumarin moieties connected through various linkers were synthesized in order to synergize the inhibition potential of both the moieties against four selected human carbonic anhydrase isoforms (hCA I, II, IX & XII). All compounds were found to be potent inhibitors of tumor associated hCA IX & XII while at the same time required large amounts to inhibit off-targeted housekeeping hCA I & II. Selectivity was more pronounced against hCA II over I, and hCA XII over IX. Results were compared with antitumor drug acetazolamide. One derivative 2b of series 2 was found to be a better selective inhibitor of hCA IX and XII. PMID:27137360

  20. Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas.

    Science.gov (United States)

    Lounnas, Nadia; Rosilio, Célia; Nebout, Marielle; Mary, Didier; Griessinger, Emmanuel; Neffati, Zouhour; Chiche, Johanna; Spits, Hergen; Hagenbeek, Thijs J; Asnafi, Vahid; Poulsen, Sally-Ann; Supuran, Claudiu T; Peyron, Jean-François; Imbert, Véronique

    2013-06-01

    The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN-/- mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL. PMID:23348702

  1. Carbonic anhydrase activators: gold nanoparticles coated with derivatized histamine, histidine, and carnosine show enhanced activatory effects on several mammalian isoforms.

    Science.gov (United States)

    Saada, Mohamed-Chiheb; Montero, Jean-Louis; Vullo, Daniela; Scozzafava, Andrea; Winum, Jean-Yves; Supuran, Claudiu T

    2011-03-10

    Lipoic acid moieties were attached to amine or amino acids showing activating properties against the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of histamine, L-histidine methyl ester, and L-carnosine methyl ester were attached to gold nanoparticles (NPs) by reaction with Au(III) salts in reducing conditions. The CA activators (CAAs)-coated NPs showed low nanomolar activation (K(A)s of 1-9 nM) of relevant cytosolic, membrane-bound, mitochondrial, and transmembrane CA isoforms, such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively activated CAs ex vivo, in whole blood experiments, with an increase of 200-280% of the CA activity. This is the first example of enzyme activation with nanoparticles and may lead to biomedical applications for conditions in which the CA activity is diminished, such as aging, Alzheimer's disease, or CA deficiency syndrome. PMID:21291238

  2. Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus

    Directory of Open Access Journals (Sweden)

    Muhammad Yousuf Ali

    2015-12-01

    Full Text Available The pH and salinity balance mechanisms of crayfish are controlled by a set of transport-related genes. We identified a set of the genes from the gill transcriptome from a freshwater crayfish Cherax quadricarinatus using the Illumina NGS-sequencing technology. We identified and characterized carbonic anhydrase (CA genes and some other key genes involved in systematic acid-base balance and osmotic/ionic regulation. We also examined expression patterns of some of these genes across different sublethal pH levels [1]. A total of 72,382,710 paired-end Illumina reads were assembled into 36,128 contigs with an average length of 800 bp. About 37% of the contigs received significant BLAST hits and 22% were assigned gene ontology terms. These data will assist in further physiological-genomic studies in crayfish.

  3. DNA cloning, characterization, and inhibition studies of an α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Isik, Semra; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Scozzafava, Andrea; Supuran, Claudiu T; Capasso, Clemente

    2012-12-13

    We have cloned, purified, and characterized an α-carbonic anhydrase (CA, EC 4.2.1.1) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity, and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which are methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, and indisulam (KI values in the range 0.69-8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered until now. PMID:23181552

  4. The extremo-α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium azorense is highly inhibited by sulfonamides.

    Science.gov (United States)

    Vullo, Daniela; De Luca, Viviana; Scozzafava, Andrea; Carginale, Vincenzo; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2013-08-01

    The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) is the most effective CA known to date. Here we investigated the inhibition profile of this enzyme with a series of aromatic and heterocyclic sulfonamides, and one sulfamate. Many clinically used sulfonamides, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, celecoxib and sulpiride were low nanomolar/subnanomolar SazCA inhibitors (KIs in the range of 0.9-10.8 nM) whereas simple aromatic derivatives were less effective as SazCA inhibitors. The inhibition profile of SazCA is slightly different from that of the related enzyme from S. yellostonense (SspCA), investigated earlier by our groups. PMID:23777827

  5. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

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    Jérôme eDoyen

    2013-01-01

    Full Text Available The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases CAIX and CAXII constitute a robust pHi-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX and LS174Tr cells (inducible knock-down for ca9/ca12 were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2 exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of NHE-1 sensitize cells to radiation-induced cell death. Secondly, the pHi-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50% and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating carbonic anhydrases as an anti-cancer strategy.

  6. Significance of different carbon forms and carbonic anhydrase activity in monitoring and prediction of algal blooms in the urban section of Jialing River, Chongqing, China.

    Science.gov (United States)

    Nie, Yudong; Zhang, Zhi; Shen, Qian; Gao, Wenjin; Li, Yingfan

    2016-05-18

    The Three Gorges Dam is one of the largest hydroelectric power plants worldwide; its reservoir was preliminarily impounded in 2003 and finally impounded to 175 m in 2012. The impoundment caused some environmental problems, such as algal blooms. Carbonic anhydrase (CA) is an important biocatalyst in the carbon utilization by algae and plays an important role in algal blooms. CA has received considerable attention for its role in red tides in oceans, but less investigation has been focused on its role in algal blooms in fresh water. In this study, the seasonal variation of water quality parameters, different carbon forms, carbonic anhydrase activity (CAA), and the algal cell density of four sampling sites in the urban section of the Jialing River were investigated from November 1, 2013 to October 31, 2014. Results indicated that CAA exhibited a positive correlation with dissoluble organic carbon (DOC), pH, and temperature, but a negative correlation with CO2 and dissoluble inorganic carbon (DIC). Algal cell density exhibited a positive correlation with flow velocity (V), pH, particulate organic carbon (POC), and CAA, a negative correlation with CO2, and a negative partial correlation with DIC. The relationship between CAA and algal cell density for the entire year can be described as cells = 23.278CAA - 42.666POC + 139.547pH - 1057.106. The algal bloom prediction model for the key control period can be described as cells = -45.895CAA + 776.103V- 29.523DOC + 14.219PIC + 35.060POC + 19.181 (2 weeks in advance) and cells = 69.200CAA + 203.213V + 4.184CO2 + 38.911DOC + 40.770POC - 189.567 (4 weeks in advance). The findings in this study demonstrate that the carbon utilization by algae is conducted by CA and provide a new method of monitoring algal cell density and predicting algal blooms. PMID:27142237

  7. Activity and stability of immobilized carbonic anhydrase for promoting CO2 absorption into a carbonate solution for post-combustion CO2 capture

    Science.gov (United States)

    Zhang, S.; Zhang, Z.; Lu, Y.; Rostam-Abadi, M.; Jones, A.

    2011-01-01

    An Integrated Vacuum Carbonate Absorption Process (IVCAP) currently under development could significantly reduce the energy consumed when capturing CO2 from the flue gases of coal-fired power plants. The biocatalyst carbonic anhydrase (CA) has been found to effectively promote the absorption of CO2 into the potassium carbonate solution that would be used in the IVCAP. Two CA enzymes were immobilized onto three selected support materials having different pore structures. The thermal stability of the immobilized CA enzymes was significantly greater than their free counterparts. For example, the immobilized enzymes retained at least 60% of their initial activities after 90days at 50??C compared to about 30% for their free counterparts under the same conditions. The immobilized CA also had significantly improved resistance to concentrations of sulfate (0.4M), nitrate (0.05M) and chloride (0.3M) typically found in flue gas scrubbing liquids than their free counterparts. ?? 2011 Elsevier Ltd.

  8. Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

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    Werner E G Müller

    Full Text Available Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC. Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative enzyme carbonic anhydrase and the (putative osteoclast-stimulating factor (OSTF, that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2 show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+-depletion condition (1 mM CaCl(2. Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes to human (osteoclast.

  9. Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O.

    Science.gov (United States)

    Tu, C K; Silverman, D N

    1985-10-01

    We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme. PMID:3936538

  10. Complexes With Biologically Active Ligands. Part 4. Coordination Compounds of Chlorothiazide With Transition Metal Ions Behave as Strong Carbonic Anhydrase Inhibitors

    OpenAIRE

    Supuran, Claudiu T.

    1996-01-01

    Complexes of the diuretic benzothiadiazine derivative chlorothiazide (6-chloro-7-sulfamoyl- 1,2,4-benzothiadiazine-1,1-dioxide) with V(IV); Fe(II); Co(II); Ni(II); Cu(II), Ag(I) and U(VI) were prepared and characterized by elemental analysis, spectroscopic, thermogravimetric, magnetic and conductimetric measurements. The complexes behave as effective inhibitors for two isozymes (I and II) of carbonic anhydrase (CA).

  11. Quantitative Characterization of the Interaction Space of the Mammalian Carbonic Anhydrase Isoforms I, II, VII, IX, XII, and XIV and their Inhibitors, Using the Proteochemometric Approach.

    Science.gov (United States)

    Rasti, Behnam; Karimi-Jafari, Mohammad H; Ghasemi, Jahan B

    2016-09-01

    The critical role of carbonic anhydrases in different physiological processes has put this protein family at the center of attention, challenging major diseases like glaucoma, neurological disorders such as epilepsy and Alzheimer's disease, obesity, and cancers. Many QSAR/QSPR (quantitative structure-activity/property relationship) researches have been carried out to design potent carbonic anhydrase inhibitors (CAIs); however, using inhibitors with no selectivity for different isoforms can lead to major side-effects. Given that QSAR/QSPR methods are not capable of covering multiple targets in a unified model, we have applied the proteochemometric approach to model the interaction space that governs selective inhibition of different CA isoforms by some mono-/dihydroxybenzoic acid esters. Internal and external validation methods showed that all models were reliable in terms of both validity and predictivity, whereas Y-scrambling assessed the robustness of the models. To prove the applicability of our models, we showed how structural changes of a ligand can affect the selectivity. Our models provided interesting information that can be useful for designing inhibitors with selective behavior toward isoforms of carbonic anhydrases, aiding in their selective inhibition. PMID:26990115

  12. Carbonic anhydrase-related protein XI: structure of the gene in the greater false vampire bat (Megaderma lyra) compared with human and domestic pig.

    Science.gov (United States)

    Porter, Calvin A; Hewett-Emmett, David; Tashian, Richard E

    2013-06-01

    Carbonic anhydrase-related protein XI (CA-RP XI) is a member of the α-carbonic anhydrase family (encoded by the gene CA-11), which has lost features of the active site required for enzymatic activity. Using PCR, we amplified CA-11 from genomic DNA of the bat Megaderma lyra. To elucidate the gene structure, we sequenced PCR products and compared their sequences with genomic and mRNA sequences known from human and domestic pig. We identified and sequenced eight introns in the bat CA-11. Five introns (introns 3-7) are located in identical or similar positions in other members of the vertebrate α-carbonic anhydrase gene family. Two 5' introns and one 3' intron are located in the regions of little or no sequence similarity with other members of the gene family. The low sequence similarity and additional introns suggest a separate evolutionary origin for the 5' and 3' portions of the CA-RP XI gene. PMID:23417223

  13. Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    Science.gov (United States)

    De Luca, Viviana; Vullo, Daniela; Del Prete, Sonia; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-02-15

    We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. PMID:26778292

  14. Synthesis of a new series of dithiocarbamates with effective human carbonic anhydrase inhibitory activity and antiglaucoma action.

    Science.gov (United States)

    Bozdag, Murat; Carta, Fabrizio; Vullo, Daniela; Akdemir, Atilla; Isik, Semra; Lanzi, Cecilia; Scozzafava, Andrea; Masini, Emanuela; Supuran, Claudiu T

    2015-05-15

    A new series of dithiocarbamates (DTCs) was prepared from primary/secondary amines incorporating amino/hydroxyl-alkyl, mono- and bicyclic aliphatic ring systems based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, and carbon disulfide. The compounds were investigated for the inhibition of four mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, that is, the human (h) hCA I, II, IX and XII, drug targets for antiglaucoma (hCA II and XII) or antitumor (hCA IX/XII) agents. The compounds were moderate or inefficient hCA I inhibitors (off-target isoform for both applications), efficiently inhibited hCA II, whereas some of them were low nanomolar/subnanomolar hCA IX/XII inhibitors. One DTC showed excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, with a two times better efficiency compared to the clinically used sulfonamide dorzolamide. PMID:25846066

  15. Identification of two carbonic anhydrases in the mantle of the European Abalone Haliotis tuberculata (Gastropoda, Haliotidae): phylogenetic implications.

    Science.gov (United States)

    LE Roy, Nathalie; Marie, Benjamin; Gaume, Béatrice; Guichard, Nathalie; Delgado, Sidney; Zanella-Cléon, Isabelle; Becchi, Michel; Auzoux-Bordenave, Stéphanie; Sire, Jean-Yves; Marin, Frédéric

    2012-07-01

    Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks.

  16. Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

    Science.gov (United States)

    De Luca, Viviana; Vullo, Daniela; Del Prete, Sonia; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-02-15

    We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat of 6.0×10(5)s(-1) and a kcat/Km of 4.7×10(6)M(-1)×s(-1). This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a KI of 502nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed KIs in the range of 1.4-4.4mM. Diethyldithiocarbamate was a better inhibitor (KI of 0.58mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with KIs ranging between 8 and 38μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.

  17. Cloning, expression, purification and sulfonamide inhibition profile of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-09-01

    We report the cloning, purification and characterization of the full domain of carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, which incorporates 358 amino acid residues (from 181 to 538, in the sequence of this 600 amino acid long protein), called PfCAdom. The enzyme, which belongs to the η-CA class showed the following kinetic parameters: kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), being 13.3 times more effective as a catalyst compared to the truncated form PfCA. PfCAdom is more effective than the human (h) isoform hCA I, being around 50% less effective compared to hCA II, one of the most catalytically efficient enzymes known so far. Intriguingly, the sulfonamides CA inhibitors generally showed much weaker inhibitory activity against PfCAdom compared to PfCA, prompting us to hypothesize that the 69 amino acid residues insertion present in the active site of this η-CA is crucial for the active site architecture. The best sulfonamide inhibitors for PfCAdom were acetazolamide, methazolamide, metanilamide and sulfanilamide, with KIs in the range of 366-808nM. PMID:27485387

  18. Anion and sulfonamide inhibition studies of an α-carbonic anhydrase from the Antarctic hemoglobinless fish Chionodraco hamatus.

    Science.gov (United States)

    Cincinelli, Alessandra; Martellini, Tania; Vullo, Daniela; Supuran, Claudiu T

    2015-12-01

    An α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from the Antarctic hemoglobinless fish Chionodraco hamatus (icefish). The new enzyme, denominated ChaCA, has a good catalytic activity for the physiologic CO2 hydration to bicarbonate reaction, similar to that of the low activity human isoform hCA I, with a kcat of 5.3×10(5) s(-1), and a kcat/Km of 3.7×10(7) M(-1) s(-1). The enzyme was inhibited in the submillimolar range by most inorganic anions (cyanate, thiocyanate, cyanide, bicarbonate, halides), whereas sulfamide, sulfamate, phenylboronic/phenylarsonic acids were micromolar inhibitors, with KIs in the range of 9-77 μM. Many clinically used drugs, such as acetazolamide, methazolamide, dorzolamide, brinzolamide, topiramate and benzolamide were low nanomolar inhibitors, with KIs in the range of 39.1-77.6 nM. As the physiology of CO2/bicarbonate transport or the Root effect in this Antarctic fish are poorly understood at this moment, such inhibition data may give a more detailed insight in the role that CAs play in these phenomena, by the use of inhibitors described here as physiologic tools. PMID:26525863

  19. A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms.

    Science.gov (United States)

    Carradori, Simone; Secci, Daniela; De Monte, Celeste; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Codispoti, Rossella; De Cosmi, Federica; Guglielmi, Paolo; Supuran, Claudiu T

    2016-03-01

    Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with KIs ranging between 19 and 2482nM, whereas they were poorly active against hCA II (KIs >10μM) and hCA I (KIs ranging between 318nM and 50μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors. PMID:26810710

  20. Sulfonamide inhibition studies of the β-carbonic anhydrase from the newly discovered bacterium Enterobacter sp. B13.

    Science.gov (United States)

    Eminoğlu, Ayşenur; Vullo, Daniela; Aşık, Aycan; Çolak, Dilşat Nigar; Çanakçı, Sabriye; Beldüz, Ali Osman; Supuran, Claudiu T

    2016-04-01

    The genome of the newly identified bacterium Enterobacter sp. B13 encodes for a β-class carbonic anhydrases (CAs, EC 4.2.1.1), EspCA. This enzyme was recently cloned, and characterized kinetically by this group (J. Enzyme Inhib. Med. Chem. 2016, 31). Here we report an inhibition study with sulfonamides and sulfamates of this enzyme. The best EspCA inhibitors were some sulfanylated sulfonamides with elongated molecules, metanilamide, 4-aminoalkyl-benzenesulfonamides, acetazolamide, and deacetylated methazolamide (KIs in the range of 58.7-96.5nM). Clinically used agents such as methazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide, zonisamide, sulthiame, sulpiride, topiramate and valdecoxib were slightly less effective inhibitors (KIs in the range of 103-138nM). Saccharin, celecoxib, dichlorophenamide and many simple benzenesulfonamides were even less effective as EspCA inhibitors, with KIs in the range of 384-938nM. Identification of effective inhibitors of this bacterial enzyme may lead to pharmacological tools useful for understanding the physiological role(s) of the β-class CAs in bacterial pathogenicity/virulence. PMID:26920803

  1. Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies.

    Science.gov (United States)

    Carta, Fabrizio; Ferraroni, Marta; Scozzafava, Andrea; Supuran, Claudiu T

    2016-01-15

    Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (KIs of 366-127 nM), CA IX (KIs of 8.1-36.9 nM), CA XII (KIs of 4.1-20.5 nM) and CA XIV (KIs of 12.8-53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies. PMID:26682703

  2. The human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibition effects of trimethoxyindane derivatives.

    Science.gov (United States)

    Taslimi, Parham; Gulcin, Ilhami; Ozgeris, Bunyamin; Goksu, Suleyman; Tumer, Ferhan; Alwasel, Saleh H; Supuran, Claudiu T

    2016-01-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) had six genetically distinct families described to date in various organisms. There are 16 known CA isoforms in humans. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. Acetylcholine esterase (AChE. EC 3.1.1.7) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine relaying the signal from the nerve. In this study, some trimethoxyindane derivatives were investigated as inhibitors against the cytosolic hCA I and II isoenzymes, and AChE enzyme. Both hCA isozymes were inhibited by trimethoxyindane derivatives in the low nanomolar range. These compounds were good hCA I inhibitors (Kis in the range of 1.66-4.14 nM) and hCA II inhibitors (Kis of 1.37-3.12 nM) and perfect AChE inhibitors (Kis in the range of 1.87-7.53 nM) compared to acetazolamide as CA inhibitor (Ki: 6.76 nM for hCA I and Ki: 5.85 nM for hCA II) and Tacrine as AChE inhibitor (Ki: 7.64 nM). PMID:25697270

  3. Sulfonamide inhibition studies of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2, TcruCA.

    Science.gov (United States)

    Vullo, Daniela; Bhatt, Avni; Mahon, Brian P; McKenna, Robert; Supuran, Claudiu T

    2016-01-15

    We report a sulfonamide/sulfamate inhibition study of the α-carbonic anhydrase (CA, EC 4.2.1.1) present in the gammaproteobacterium Thiomicrospira crunogena XCL-2, a mesophilic hydrothermal vent-isolate organism, TcruCA. As Thiomicrospira crunogena is one of thousands of marine organisms that uses CA for metabolic regulation, the effect of sulfonamide inhibition has been considered. Sulfonamide-based drugs have been widely used in a variety of antibiotics, and bioelimination of these compounds results in exposure of these compounds to marine life. The enzyme was highly inhibited, with Ki values ranging from 2.5 to 40.7nM by a variety of sulfonamides including acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide and benzenesulfonamides incorporating 4-hydroxyalkyl moieties. Less effective inhibitors were topiramate, zonisamide, celecoxib, saccharin and hydrochlorothiazide as well as simple benzenesulfonamides incorporating amino, halogeno, alkyl, aminoalkyl and other moieties in the ortho- or para-positions of the aromatic ring (Kis of 202-933nM). The active site interactions between TcruCA and three clinically-used CA inhibitors, acetazolamide (Diamox®), dorzolamide (Trusopt®), and brinzolamide (Azopt®) are studied using molecular docking to provide insight into the reported Ki values. Comparison between various enzymes belonging to this family may also bring interesting hints in these fascinating phenomena. PMID:26691758

  4. Carbonic anhydrase inhibitors: Design, synthesis, kinetic, docking and molecular dynamics analysis of novel glycine and phenylalanine sulfonamide derivatives.

    Science.gov (United States)

    Fidan, İsmail; Salmas, Ramin Ekhteiari; Arslan, Mehmet; Şentürk, Murat; Durdagi, Serdar; Ekinci, Deniz; Şentürk, Esra; Coşgun, Sedat; Supuran, Claudiu T

    2015-12-01

    The inhibition of two human cytosolic carbonic anhydrase isozymes I and II, with some novel glycine and phenylalanine sulfonamide derivatives were investigated. Newly synthesized compounds G1-4 and P1-4 showed effective inhibition profiles with KI values in the range of 14.66-315μM for hCA I and of 18.31-143.8μM against hCA II, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico docking studies were applied. Atomistic molecular dynamic simulations were performed for docking poses which utilize to illustrate the inhibition mechanism of used inhibitors into active site of CAII. These sulfonamide containing compounds generally were competitive inhibitors with 4-nitrophenylacetate as substrate. Some investigated compounds here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide, sulfanilamide or mafenide and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

  5. 植物碳酸酐酶的研究进展%Progress in Research on Plant Carbonic Anhydrase

    Institute of Scientific and Technical Information of China (English)

    蒋春云; 马秀灵; 沈晓艳; 李燕; 赵彦修

    2013-01-01

    在植物组织中,碳酸酐酶(CA)催化CO2与HCO3-之间可逆的水合反应,重新固定呼吸释放的CO2并用于细胞光合作用.本文简要介绍了CA的生理机能、分类、亚细胞定位、基因功能等的研究进展,并展望了CA在提高C3植物光合效率以及CA在C3植物由C3光合类型转向C4光合类型方面的研究意义.%Carbonic anhydrase (CA) catalyses the reversible reaction between CO2 and HCO3-in plant living organisms.It can refix the respiration-released CO2 which participates in photosynthesis process.In this article we summarize the research progress in the physiological function,classification,subcellular localization and gene function of CA.And we prospect its crucial roles in increasing the photosynthetic rate in C3 plants and in the type of photosynthesis from C3 to C4.

  6. Evaluation of a Carbonic Anhydrase IX-Targeted Near-Infrared Dye for Fluorescence-Guided Surgery of Hypoxic Tumors.

    Science.gov (United States)

    Lv, Peng-Cheng; Roy, Jyoti; Putt, Karson S; Low, Philip S

    2016-05-01

    Proof-of-principle studies in ovarian, lung, and brain cancer patients have shown that fluorescence-guided surgery can enable removal of otherwise undetectable malignant lesions, decrease the number of cancer-positive margins, and permit identification of disease-containing lymph nodes that would have normally evaded resection. Unfortunately, the current arsenal of tumor-targeted fluorescent dyes does not permit identification of all cancers, raising the need to design new tumor-specific fluorescent dyes to illuminate the currently undetectable cancers. In an effort to design a more universal fluorescent cancer imaging agent, we have undertaken to synthesize a fluorophore that could label all hypoxic regions of tumors. We report here the synthesis, in vitro binding, and in vivo imaging of a near-infrared (NIR) fluorescent dye that is targeted to carbonic anhydrase IX (CA IX), i.e., a widely accepted marker of hypoxic tissues. The low molecular weight NIR probe, named Hypoxyfluor, is shown to bind CA IX with high affinity and accumulate rapidly and selectively in CA IX positive tumors. Because nearly all human cancers contain hypoxic regions that express CA IX abundantly, this NIR probe should facilitate surgical resection of a wide variety of solid tumors. PMID:27043317

  7. Self-healing of Early Age Cracks in Cement-based Materials by Mineralization of Carbonic Anhydrase Microorganism

    Directory of Open Access Journals (Sweden)

    Chunxiang eQian

    2015-11-01

    Full Text Available This research investigated the self-healing potential of early age cracks in cement-based materials incorporating the bacteria which can produce carbonic anhydrase. Cement-based materials specimens were pre-cracked at the age of 7, 14, 28, 60 days to study the repair ability influenced by cracking time, the width of cracks were between 0.1 and 1.0 mm to study the healing rate influenced by width of cracks. The experimental results indicated that the bacteria showed excellent repairing ability to small cracks formed at early age of 7 days, cracks below 0.4 mm was almost completely closed. The repair effect reduced with the increasing of cracking age. Cracks width influenced self-healing effectiveness significantly. The transportation of CO2 and Ca2+ controlled the self-healing process. The computer simulation analyses revealed the self-healing process and mechanism of microbiologically precipitation induced by bacteria and the depth of precipitated CaCO3 could be predicted base on valid Ca2+.

  8. Intrinsic thermodynamics of 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamide binding to carbonic anhydrases by isothermal titration calorimetry.

    Science.gov (United States)

    Zubrienė, Asta; Smirnovienė, Joana; Smirnov, Alexey; Morkūnaitė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Juozapaitienė, Vaida; Norvaišas, Povilas; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

    2015-10-01

    Para substituted tetrafluorobenzenesulfonamides bind to carbonic anhydrases (CAs) extremely tightly and exhibit some of the strongest known protein-small ligand interactions, reaching an intrinsic affinity of 2 pM as determined by displacement isothermal titration calorimetry (ITC). The enthalpy and entropy of binding to five CA isoforms were measured by ITC in two buffers of different protonation enthalpies. The pKa values of compound sulfonamide groups were measured potentiometrically and spectrophotometrically, and enthalpies of protonation were measured by ITC in order to evaluate the proton linkage contributions to the observed binding thermodynamics. Intrinsic means the affinity of a sulfonamide anion for the Zn bound water form of CAs. Fluorination of the benzene ring significantly enhanced the observed affinities as it increased the fraction of deprotonated ligand while having little impact on intrinsic affinities. Intrinsic enthalpy contributions to the binding affinity were dominant over entropy and were more exothermic for CA I than for other CA isoforms. Thermodynamic measurements together with the X-ray crystallographic structures of protein-ligand complexes enabled analysis of structure-activity relationships in this enzyme ligand system.

  9. Impacts of Elevated CO2 Concentration on Biochemical Composition,Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

    Institute of Scientific and Technical Information of China (English)

    Jian-Rong XIA; Kun-Shan GAO

    2005-01-01

    To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations,Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2concentrations within the range 3-186 μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

  10. Carbonic Anhydrase VI Gene Polymorphism rs2274327 Relationship Between Salivary Parameters and Dental-Oral Health Status in Children.

    Science.gov (United States)

    Sengul, Fatih; Kilic, Munevver; Gurbuz, Taskin; Tasdemir, Sener

    2016-08-01

    The aim of this study was to research carbonic anhydrase (CA) VI one single-nucleotide polymorphism (SNP) and its potential association with dental-oral health status (dental caries, Plaque Index (PI) and Gingival Index (GI)) and salivary parameters (salivary buffering capacity, salivary flow rate (SFR)) in children. A total of 178 children were divided into two groups: non-carious (n = 70, 34 boys and 36 girls) and carious (n = 108, 47 boys and 61 girls). The clinical evaluations were performed according to the decayed, missing, and filled teeth (dmft/DMFT) index by a specialist. Clinical parameters including PI, GI, and simplified oral hygiene index (OHI-S) were recorded. Salivary pH (SpH) was measured using pH paper. Blood samples and unstimulated whole saliva were collected, and SFR was calculated. The CA VI rs2274327 polymorphism was determined by a LightSNiP assay on the realtime PCR system. The frequencies of rs2274327 were not significant between groups (p > 0.05). There was a positive correlation between OHI-S and SpH in the carious and non-carious groups (p OHI-S, PI, GI, SFR, and SpH (p > 0.05). CA VI SNP (rs2274327) had no statistically significant association with OHI-S, PI, GI, SFR, and SpH in the children.

  11. Carbonic Anhydrase VI Gene Polymorphism rs2274327 Relationship Between Salivary Parameters and Dental-Oral Health Status in Children.

    Science.gov (United States)

    Sengul, Fatih; Kilic, Munevver; Gurbuz, Taskin; Tasdemir, Sener

    2016-08-01

    The aim of this study was to research carbonic anhydrase (CA) VI one single-nucleotide polymorphism (SNP) and its potential association with dental-oral health status (dental caries, Plaque Index (PI) and Gingival Index (GI)) and salivary parameters (salivary buffering capacity, salivary flow rate (SFR)) in children. A total of 178 children were divided into two groups: non-carious (n = 70, 34 boys and 36 girls) and carious (n = 108, 47 boys and 61 girls). The clinical evaluations were performed according to the decayed, missing, and filled teeth (dmft/DMFT) index by a specialist. Clinical parameters including PI, GI, and simplified oral hygiene index (OHI-S) were recorded. Salivary pH (SpH) was measured using pH paper. Blood samples and unstimulated whole saliva were collected, and SFR was calculated. The CA VI rs2274327 polymorphism was determined by a LightSNiP assay on the realtime PCR system. The frequencies of rs2274327 were not significant between groups (p > 0.05). There was a positive correlation between OHI-S and SpH in the carious and non-carious groups (p  0.05). CA VI SNP (rs2274327) had no statistically significant association with OHI-S, PI, GI, SFR, and SpH in the children. PMID:27100223

  12. Synthesis and carbonic anhydrase inhibitory properties of amino acid - coumarin/quinolinone conjugates incorporating glycine, alanine and phenylalanine moieties.

    Science.gov (United States)

    Küçükbay, F Zehra; Küçükbay, Hasan; Tanc, Muhammet; Supuran, Claudiu T

    2016-12-01

    N-Protected amino acids (Gly, Ala and Phe) were reacted with amino substituted coumarin and quinolinone derivatives, leading to the corresponding N-protected amino acid-coumarin/quinolinone conjugates. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against various human (h) isoforms, such as hCA I, hCA II, hCA IV and hCA XII. The quinolinone conjugates were inactive as enzyme inhibitors, whereas the coumarins were ineffective hCA I/II inhibitors (KIs > 50 μM) but were submicromolar hCA IV and XII inhibitors, with inhibition constants ranging between 92 nM and 1.19 μM for hCA IV, and between 0.11 and 0.79 μM for hCA XII. These coumarin derivatives, as many others reported earlier, thus show an interesting selective inhibitory profile for the membrane-bound over the cytosolic CA isoforms.

  13. Effects of intraleaf variations in carbonic anhydrase activity and gas exchange on leaf C18OO isoflux in Zea mays.

    Science.gov (United States)

    Affek, Hagit P; Krisch, Maria J; Yakir, Dan

    2006-01-01

    Variation in the C18OO content of atmospheric CO2 (delta18Oa) can be used to distinguish photosynthesis from soil respiration, which is based on carbonic anhydrase (CA)-catalyzed 18O exchange between CO2 and 18O-enriched leaf water (delta18Ow). Here we tested the hypothesis that mean leaf delta18Ow and assimilation rates can be used to estimate whole-leaf C18OO flux (isoflux), ignoring intraleaf variations in CA activity and gas exchange parameters. We observed variations in CA activity along the leaf (> 30% decline from the leaf center toward the leaf ends), which were only partially correlated to those in delta18Ow (7 to 21 per thousand), delta18O and delta13C of leaf organic matter (25 to 30 per thousand and -12.8 to -13.2 per thousand, respectively), and substomatal CO2 concentrations (intercellular CO2 concentrations, c(i), at the leaf center were approximately 40% of those at the leaf tip). The combined effect of these variations produced a leaf-integrated isoflux that was different from that predicted based on bulk leaf values. However, because of canceling effects among the influencing parameters, isoflux overestimations were only approximately 10%. Conversely, use of measured parameters from a leaf segment could produce large errors in predicting leaf-integrated C18OO fluxes. PMID:16411935

  14. Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide.

    Directory of Open Access Journals (Sweden)

    Shenghua Huang

    Full Text Available The β-class carbonic anhydrases (β-CAs are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion--a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2-HCO(3(- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.

  15. Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide.

    Science.gov (United States)

    Huang, Shenghua; Hainzl, Tobias; Grundström, Christin; Forsman, Cecilia; Samuelsson, Göran; Sauer-Eriksson, A Elisabeth

    2011-01-01

    The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion--a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3)(-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules. PMID:22162771

  16. Epidermal carbonic anhydrase activity and exoskeletal metal content during the molting cycle of the blue crab, Callinectes sapidus.

    Science.gov (United States)

    Calhoun, Stacy; Zou, Enmin

    2016-03-01

    During the crustacean molting cycle, the exoskeleton is first mineralized in postmolt and intermolt and then presumably demineralized in premolt in order for epidermal retraction to occur. The mineralization process calls for divalent metal ions, such as Ca(2+) and Mg(2+) , and bicarbonate ions whereas protons are necessary for dissolution of carbonate salts. Carbonic anhydrase (CA) has been suggested to be involved in exoskeletal mineralization by providing bicarbonate ions through catalyzing the reaction of carbon dioxide hydration. However, results of earlier studies on the role of epidermal CA in metal incorporation in crustacean exoskeleton are not consistent. This study was aimed to provide further evidence to support the notion that epidermal CA is involved in exoskeletal mineralization using the blue crab, Callinectes sapidus (Rathbun 1896), as the model crustacean. Significant increases first in calcium and magnesium then in manganese post-ecdysis indicate significant metal deposition during postmolt and intermolt. Significant positive correlation between calcium or magnesium content and epidermal CA activity in postmolt and intermolt constitutes evidence that CA is involved in the mineralization of the crustacean exoskeleton. Additionally, we proposed a hypothetical model to describe the role of epidermal CA in both mineralization and demineralization of the exoskeleton based on the results of epidermal CA activity and exoskeletal metal content during the molting cycle. Furthermore, we found that the pattern of epidermal CA activity during the molting cycle of C. sapidus is similar to that of ecdysteroids reported for the same species, suggesting that epidermal CA activity may be under control of the molting hormones. PMID:26935248

  17. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors

    Directory of Open Access Journals (Sweden)

    Olga eSedlakova

    2014-01-01

    Full Text Available Acidic tissue microenvironment contributes to tumor progression via multiple effects including the activation of angiogenic factors and proteases, reduced cell-cell adhesion, increased migration and invasion, etc. In addition, intratumoral acidosis can influence the uptake of anticancer drugs and modulate the response of tumors to conventional therapy. Acidification of the tumor microenvironment often develops due to hypoxia-triggered oncogenic metabolism, which leads to the extensive production of lactate, protons and carbon dioxide. In order to avoid intracellular accumulation of the acidic metabolic products, which is incompatible with the survival and proliferation, tumor cells activate molecular machinery that regulates pH by driving transmembrane inside-out and outside-in ion fluxes. Carbonic anhydrase IX (CA IX is a hypoxia-induced catalytic component of the bicarbonate import arm of this machinery. Through its catalytic activity, CA IX directly participates in many acidosis-induced features of tumor phenotype as demonstrated by manipulating its expression and/or by in vitro mutagenesis. CA IX can function as a survival factor protecting tumor cells from hypoxia and acidosis, as a pro-migratory factor facilitating cell movement and invasion, as a signaling molecule transducing extracellular signals to intracellular pathways (including major signaling and metabolic cascades and converting intracellular signals to extracellular effects on adhesion, proteolysis and other processes. These functional implications of CA IX in cancer are supported by numerous clinical studies demonstrating the association of CA IX with various clinical correlates and markers of aggressive tumor behavior. Although our understanding of the many faces of CA IX is still incomplete, existing knowledge supports the view that CA IX is a biologically and clinically relevant molecule, exploitable in anticancer strategies aimed at targeting adaptive responses to hypoxia

  18. Oxygen-18 Exchange as a Measure of Accessibility of CO2 and HCO3− to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263) 1

    Science.gov (United States)

    Tu, C. K.; Acevedo-Duncan, Mildred; Wynns, George C.; Silverman, David N.

    1986-01-01

    We have measured the exchange of 18O between CO2 and H2O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of 18O from CO2 in the fluid outside the cells provides a method to study CO2 and HCO3− kinetics in suspensions of algae that contain carbonic anhydrase since 18O loss to H2O is catalyzed inside the cells but not in the external fluid. Low-CO2 cells of Chlorella vulgaris (grown with air) were added to a solution containing 18O enriched CO2 and HCO3− with 2 to 15 millimolar total inorganic carbon. The observed depletion of 18O from CO2 was biphasic and the resulting 18C content of CO2 was much less than the 18O content of HCO3− in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO3− into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10−4 s−1 due to our experimental errors. The Fick's law rate constant for entry of CO2 to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO2 flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO2 entry into the cell or both. The CO2 hydration activity inside the cells was 160 times the uncatalyzed CO2 hydration rate. PMID:16664755

  19. Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263).

    Science.gov (United States)

    Tu, C K; Acevedo-Duncan, M; Wynns, G C; Silverman, D N

    1986-04-01

    We have measured the exchange of (18)O between CO(2) and H(2)O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of (18)O from CO(2) in the fluid outside the cells provides a method to study CO(2) and HCO(3) (-) kinetics in suspensions of algae that contain carbonic anhydrase since (18)O loss to H(2)O is catalyzed inside the cells but not in the external fluid. Low-CO(2) cells of Chlorella vulgaris (grown with air) were added to a solution containing (18)O enriched CO(2) and HCO(3) (-) with 2 to 15 millimolar total inorganic carbon. The observed depletion of (18)O from CO(2) was biphasic and the resulting (18)C content of CO(2) was much less than the (18)O content of HCO(3) (-) in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO(3) (-) into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10(-4) s(-1) due to our experimental errors. The Fick's law rate constant for entry of CO(2) to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO(2) flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO(2) entry into the cell or both. The CO(2) hydration activity inside the cells was 160 times the uncatalyzed CO(2) hydration rate. PMID:16664755

  20. Oxygen-18 exchange as a measure of accessibility of CO2 and HCO3- to carbonic anhydrase in Chlorella vulgaris (UTEX 263)

    International Nuclear Information System (INIS)

    The exchange of 18O between CO2 and H2O in stirred suspensions of Chlorella vulgaris (UTEX 263) was measured using a membrane inlet to a mass spectrometer. The depletion of 18O from CO2 in the fluid outside the cells provides a method to study CO2 and HCO3- kinetics in suspensions of algae that contain carbonic anhydrase since 18O loss to H2O is catalyzed inside the cells but not in the external fluid. Low-CO2 cells of Chlorella vulgaris (grown with air) were added to a solution containing 18O enriched CO2 and HCO3- with 2 to 15 millimolar total inorganic carbon. The observed depletion of 18O from CO2 was biphasic and the resulting 18O content of CO2 was much less than the 18O content of HCO3- in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO3- into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10-4 s-1 due to experimental errors. The Fick's law rate constant for entry of CO2 to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO2 flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO2 entry into the cell or both. The CO2 hydration activity inside the cells was 160 times the uncatalyzed CO2 hydration rate

  1. Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

    Science.gov (United States)

    Zhou, Wei; Sui, Zhenghong; Wang, Jinguo; Hu, Yiyi; Kang, Kyoung Ho; Hong, Hye Ran; Niaz, Zeeshan; Wei, Huihui; Du, Qingwei; Peng, Chong; Mi, Ping; Que, Zhou

    2016-06-01

    There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P 336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae.

  2. Effects of sodium bicarbonate concentration on growth, photosynthesis, and carbonic anhydrase activity of macroalgae Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae (Gracilariales, Rhodophyta).

    Science.gov (United States)

    Zhou, Wei; Sui, Zhenghong; Wang, Jinguo; Hu, Yiyi; Kang, Kyoung Ho; Hong, Hye Ran; Niaz, Zeeshan; Wei, Huihui; Du, Qingwei; Peng, Chong; Mi, Ping; Que, Zhou

    2016-06-01

    There is potential for bicarbonate to improve crop yields and economic efficiency of marine algae. However, few studies have focused on the effect of bicarbonate on the growth, photosynthesis, and enzyme activity associated with carbon utilization, especially in commercial macroalgae. Here, the addition of bicarbonate (up to 420 mg L(-1)) to macroalgal cultures has been evaluated for Gracilariopsis lemaneiformis, Gracilaria vermiculophylla, and Gracilaria chouae with respect to growth rate, photosynthetic activity, carbonic anhydrase activity, and biochemical composition. The results showed that the effects of NaHCO3 on growth, chlorophyll a, phycoerythrin, photosynthetic oxygen evolution, photochemical parameters of PSI and PSII, carbonic anhydrase activity, and nitrogen content were significant (P 336 mg L(-1) for Gp. lemaneiformis and >420 mg L(-1) for the other two species). Moreover, species-specific differences induced by supplementation with bicarbonate were discovered during culture. Optimal concentrations of NaHCO3 used in this study were 252 mg L(-1) for Gp. lemaneiformis and 336 mg L(-1) for G. vermiculophylla and G. chouae. These results suggest that an adequate supplementation of sodium bicarbonate is a viable strategy for promoting growth and photosynthetic activity in some macroalgae as well as for improving biochemical composition. The study will help to accelerate the growth rate of algae and improve the quality of thalli, and will also be useful for enhancing the understanding of carbon utilization in macroalgae. PMID:26960545

  3. 呋塞米对碳酸酐酶的抑制效应再研究%Inhibitory effect of furosemide on carbonic anhydrase

    Institute of Scientific and Technical Information of China (English)

    袁美华; 蒋彦; 杨毅

    2013-01-01

    The inhibitory effect of a high efficient diuretic ,furosemide ,on carbonic anhydrase was investigated in this study .Compared with acetazolamide ,furosemide can quickly make BCAⅡ inactive when its concentration is close to the enzyme concentration . The results show that furosemide is a non-competitive inhibitor of carbonic anhydrase ,the vaules of its IC50 and KI are 0 .759 μM ,0 .51 μM . Acetazolamide is a competitive inhibitor of carbonic anhydrase ,the vaules of its IC5 0 and KI are 0.199μM ,0 .099 μM .%呋塞米是一种高效利尿剂,本实验主要探究其对碳酸酐酶的抑制效应.相比较乙酰唑胺而言,呋塞米在其浓度接近碳酸酐酶浓度时能使该酶基本失活.研究发现,呋塞米对碳酸酐酶的抑制效应表现为非竞争性抑制,其 IC50为0.759μM ,KI 为0.61μM ,乙酰唑胺的 IC50为0.199μM , KI 为0.099μM ,表现为竞争性抑制.

  4. Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the β-carbonic anhydrase family enzymes.

    Science.gov (United States)

    Ogawa, Takahiro; Noguchi, Keiichi; Saito, Masahiko; Nagahata, Yoshiko; Kato, Hiromi; Ohtaki, Akashi; Nakayama, Hiroshi; Dohmae, Naoshi; Matsushita, Yasuhiko; Odaka, Masafumi; Yohda, Masafumi; Nyunoya, Hiroshi; Katayama, Yoko

    2013-03-13

    Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO2 hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS. PMID:23406161

  5. Anion inhibition profiles of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-08-15

    Among the numerous metalloenzymes known to date, carbonic anhydrase (CA, EC 4.2.1.1) was the first zinc containing one, being discovered decades ago. CA is a hydro-lyase, which catalyzes the following hydration-dehydration reaction: CO2+H2O⇋HCO3(-)+H(+). Several CA classes are presently known, including the α-, β-, γ-, δ-, ζ- and η-CAs. In prokaryotes, the existence of genes encoding CAs from at least three classes (α-, β- and γ-class) suggests that these enzymes play a key role in the physiology of these organisms. In many bacteria CAs are essential for the life cycle of microbes and their inhibition leads to growth impairment or growth defects of the pathogen. CAs thus started to be investigated in detail in bacteria, fungi and protozoa with the aim to identify antiinfectives with a novel mechanism of action. Here, we investigated the catalytic activity, biochemical properties and anion inhibition profiles of the three CAs from the bacterial pathogen Vibrio cholera, VchCA, VchCAβ and VchCAγ. The three enzymes are efficient catalysts for CO2 hydration, with kcat values ranging between (3.4-8.23)×10(5)s(-1) and kcat/KM of (4.1-7.0)×10(7)M(-1)s(-1). A set of inorganic anions and small molecules was investigated for inhibition of these enzymes. The most potent VchCAγ inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with KI values ranging between 44 and 91μM. PMID:27283786

  6. Thermodynamics of binding of a sulfonamide inhibitor to metal-mutated carbonic anhydrase as studied by affinity capillary electrophoresis.

    Science.gov (United States)

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko

    2015-09-01

    By affinity capillary electrophoresis (ACE), the thermodynamic binding constants of a sulfonamide (SA) inhibitor to bovine carbonic anhydrase II (CA) and metal mutated variants (M-CAs) were evaluated. 1-(4-Aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate was used as the SA in the electrophoretic buffer for ACE. The Scatchard analysis of the dependence of the electrophoretic mobility of native CA on the SA concentration provided the binding constant to be Kb=(2.29±0.05)×10(6) M(-1) (at pH8.4, 25°C). On the other hand, apoCA showed far smaller value [Kb=(3.76±0.14)×10(2) M(-1)], suggesting that the coordination of SA to the Zn(II) center controlled the binding thermodynamics. The ACE of M-CAs showed the same behaviors as native CA but with different Kb values. For example, Co-CA adopting the same tetrahedral coordination geometry as native CA exhibited the largest Kb value [(2.55±0.05)×10(6) M(-1)] among the M-CAs. In contrast, Mn- and Ni-CA, which adopted the octahedral coordination geometry, had Kb values that were about two orders of magnitude lower. Because the hydrophobic cavity of CA around the active center pre-organized the orientation of SA, thereby fixing the ligating NH(-) moiety to the apex of the tetrahedron supported by three basal His3 of CA, metals such as Zn and Co at the center of M-CA gave the most stable CA-SA complex. However, pre-organization was not favored for octahedral geometry. Thus, pre-organization of SA was the key to facilitating the tetrahedral coordination geometry of the Zn(II) active center of CA.

  7. In vivo imaging and quantification of carbonic anhydrase IX expression as an endogenous biomarker of tumor hypoxia.

    Directory of Open Access Journals (Sweden)

    Bagna Bao

    Full Text Available Carbonic anhydrase IX (CA IX is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR fluorescent derivative of the CA IX inhibitor acetazolamide (AZ. The agent (HS680 showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%-70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231, as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6-24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8% atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo, and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.

  8. Estrogen and progesterone differentially regulate carbonic anhydrase II, III, IX, XII, and XIII in ovariectomized rat uteri.

    Science.gov (United States)

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-01-01

    Changes in the uterus expression of carbonic anhydrase (CA) II, III, IX, XII, and XIII were investigated under the influence of sex-steroids in order to elucidate mechanisms underlying differential effects of these hormones on uterine pH. Uteri of ovariectomised rats receiving over three days either vehicle, estrogen, or progesterone or three days estrogen followed by three days either vehicle or progesterone were harvested. Messenger RNA (mRNA) and protein levels were quantified by real-time PCR and Western blotting, respectively. The distribution of CA isoenzymes proteins were examined by immunohistochemistry. The levels of CAII, III, XII, and XIII mRNAs and proteins were elevated while levels of CAIX mRNA and protein were reduced following progesterone-only and estrogen plus progesterone treatment, compared to the control and estrogen plus vehicle, respectively. Following estrogen treatment, expression of CAII, IX, XII, and CAXIII mRNAs and proteins were reduced, but remained at a level higher than control, except for CAIX, where its level was higher than the control and following progesterone treatment. Under progesterone-only and estrogen plus progesterone influences, high levels of CAII, III, XII, and XIII were observed in uterine lumenal and glandular epithelia and myometrium. However, a high level of CAIX was observed only under the influence of estrogen at the similar locations. In conclusion, high expression of CAII, III, XII, and XIII under the influence of progesterone and estrogen plus progesterone could result in the reduction of uterine tissue and fluid pH; however, the significance of high levels of CAIX expression under the influence of estrogen remains unclear. PMID:26709452

  9. Potentiation of the effect of thiazide derivatives by carbonic anhydrase inhibitors: molecular mechanisms and potential clinical implications.

    Directory of Open Access Journals (Sweden)

    Kamyar Zahedi

    Full Text Available BACKGROUND: Carbonic anhydrase inhibitors (CAI are mild diuretics, hence not widely used in fluid overloaded states. They are however the treatment of choice for certain non-kidney conditions. Thiazides, specific inhibitors of Na-Cl cotransport (NCC, are mild agents and the most widely used diuretics in the world for control of mild hypertension. HYPOTHESIS: In addition to inhibiting the salt reabsorption in the proximal tubule, CAIs down-regulate pendrin, therefore leaving NCC as the major salt absorbing transporter in the distal nephron, and hence allowing for massive diuresis by the inhibitors of NCC in the setting of increased delivery of salt from the proximal tubule. EXPERIMENTAL PROTOCOLS AND RESULTS: Daily treatment of rats with acetazolamide (ACTZ, a known CAI, for 10 days caused mild diuresis whereas daily treatment with hydrochlorothiazide (HCTZ for 4 days caused hardly any diuresis. However, treatment of rats that were pretreated with ACTZ for 6 days with a combination of ACTZ plus HCTZ for 4 additional days increased the urine output by greater than 2 fold (p<0.001, n = 5 compared to ACTZ-treated animals. Sodium excretion increased by 80% in the ACTZ plus HCTZ group and animals developed significant volume depletion, metabolic alkalosis and pre-renal failure. Molecular studies demonstrated ∼75% reduction in pendrin expression by ACTZ. The increased urine output in ACTZ/HCTZ treated rats was associated with a significant reduction in urine osmolality and reduced membrane localization of AQP-2 (aquaporin2. CONCLUSIONS: These results indicate that ACTZ down-regulates pendrin expression and leaves NCC as the major salt absorbing transporter in the distal nephron in the setting of increased delivery of salt from the proximal tubule. Despite being considered mild agents individually, we propose that the combination of ACTZ and HCTZ is a powerful diuretic regimen.

  10. Metal Complexes of 1,3,4-Thiadiazole-2,5-Disulfonamide are Strong Dual Carbonic Anhydrase Inhibitors, although the Ligand Possesses very Weak such Properties

    Science.gov (United States)

    Supuran, Claudiu T.

    1995-01-01

    Coordination compounds of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with 1,3,4-thiadiazole-2,5-disulfonamide as ligand were synthesized and characterized by IR and UV spectroscopy, conductimetry and thermogravimetry. The parent ligand is a very weak carbonic anhydrase (CA) inhibitor, although it constituted the lead for developing important classes of diuretics. The complex derivatives behave as much stronger CA inhibitors, with IC50 values around 10−8M against isozyme CA II, and 10−7 M against isozyme CAI. PMID:18472784

  11. Metal Complexes of 1,3,4-Thiadiazole-2,5-Disulfonamide are Strong Dual Carbonic Anhydrase Inhibitors, although the Ligand Possesses very Weak such Properties.

    Science.gov (United States)

    Supuran, C T

    1995-01-01

    Coordination compounds of Co(II), Ni(II), Cu(II), Zn(II), and Cd(II) with 1,3,4-thiadiazole-2,5-disulfonamide as ligand were synthesized and characterized by IR and UV spectroscopy, conductimetry and thermogravimetry. The parent ligand is a very weak carbonic anhydrase (CA) inhibitor, although it constituted the lead for developing important classes of diuretics. The complex derivatives behave as much stronger CA inhibitors, with IC(50) values around 10(-8)M against isozyme CA II, and 10(-7) M against isozyme CAI.

  12. Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Ferraroni, Marta; Del Prete, Sonia; Vullo, Daniela; Capasso, Clemente; Supuran, Claudiu T

    2015-12-01

    Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA from Vibrio cholerae (VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 × 10(5) s(-1) and a kcat/Km of 4.1 × 10(7) M(-1) s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Ki of 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.

  13. Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

    NARCIS (Netherlands)

    Burghout, Peter; Cron, Lorelei E.; Gradstedt, Henrik; Quintero, Beatriz; Simonetti, Elles; Bijlsma, Jetta J. E.; Bootsma, Hester J.; Hermans, Peter W. M.

    2010-01-01

    The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO(2)) it encounters during transmission, colonization, and infection. Since CO(2) is important for various cellular processes, factors that allow optimal CO(2) sequestering are likely t

  14. Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

    Science.gov (United States)

    Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

    2009-10-01

    Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

  15. MODELLING THE INHIBITORY ACTIVITY ON CARBONIC ANHYDRASE IV OF SUBSTITUTED THIADIAZOLE - AND THIADIAZOLINE - DISULFONAMIDES: INTEGRATION OF STRUCTURE INFORMATION

    Directory of Open Access Journals (Sweden)

    Sorana Daniela Bolboaca

    2006-07-01

    Full Text Available ABSTRACT:Purpose: To analyze the relationships between inhibitory activities on carbonic anhydrase IV and structures of substituted 1,3,4-thiadiazole and 1,3,4-thiadiazoline disulfonamide through integration of compounds complex structure information by the use of Molecular Descriptors Family.Method: A number of forty compounds were used to generate and compute the molecular descriptors family and to build structure-activity relationships models. The obtained multi-varied models (the models with two, respectively with four descriptors were validated by computing the cross-validation leave-one-out score (r2cv-loo, and analyzed through assessment of the squared correlation coefficients (r2, and the models stability (r2 - r2cv-loo. The estimation abilities of the multi-varied MDF-SAR model with four descriptors were analyzed in training and test sets.Results: Analysis of the obtained models shows that the best results was obtained by the multi-varied model with four molecular descriptors (r2 = 0.920. The prediction abilities of this model is sustained by the cross validation leave-one-out score (r2cv-loo = 0.903, the model stability (r2 - r2cv-loo = 0.017, and the results on training versus test analysis (no significant differences between correlation coefficients in training and test sets, p > 0.05. The multi-varied model which used four descriptors proved to render higher value of correlation coefficient comparing with previous reported models (p 0.05. El modelo multivariante que utilizó cuatro descriptores mostró un valor más alto del coeficiente de correlación en comparación con los modelos divulgados anteriormente (p < 0.01.Conclusión: El modelo multivariante con cuatro descriptores es sólido y fiable e indica que la actividad de la inhibición en la carboanhidrasa IV producida por las sufonamidas sustituidas del 1,3,4-tiadiazol- y de la 1,3,4-tiadiazolina- dependen de la naturaleza de la geometría y de la topología del compuesto

  16. Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase Ⅱ inhibitor-induced hepatocellular carcinoma cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Su-jong YU; Hyo-suk LEE; Jung-hwan YOON; Jeong-hoon LEE; Sun-jung MYUNG; Eun-sun JANG; Min-sun KWAK; Eun-ju CHO; Ja-june JANG; Yoon-jun KIM

    2011-01-01

    Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-Ⅸ (CA-Ⅸ) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-Ⅸ in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients.Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-Ⅸ-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase Ⅱ inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array.Results: Incubation of HCC cells under hypoxia (1% 02, 5% C02, 94% N2) for 36 h significantly increased CA-IX expression level. CAI#1(400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-Ⅸ intensity was inversely related to E-cadherin intensity.Conclusion: Inhibition of hypoxia-induced CA-Ⅸ enhances hexokinase Ⅱ inhibitor-induced HCC apoptosis. Furthermore, CA-IX expres sion profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-Ⅸ, in combination with a hexokinase Ⅱ inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.

  17. Production, purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum.

    Science.gov (United States)

    Liu, Zhu; Bartlow, Patrick; Dilmore, Robert M; Soong, Yee; Pan, Zhiwei; Koepsel, Richard; Ataai, Mohammad

    2009-01-01

    Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO2. A new concept for CO2 capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO2 to HCO3(-) and H+. Cost-efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA-based CO2 capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His6 tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO2 from flue gasses. PMID:19224556

  18. Oxygen-18 exchange as a measure of accessibility of CO/sub 2/ and HCO/sub 3//sup -/ to carbonic anhydrase in Chlorella vulgaris (UTEX 263)

    Energy Technology Data Exchange (ETDEWEB)

    Tu, C.K.; Acevedo-Duncan, M.; Wynns, G.C.; Silverman, D.N.

    1986-04-01

    The exchange of /sup 18/O between CO/sub 2/ and H/sub 2/O in stirred suspensions of Chlorella vulgaris (UTEX 263) was measured using a membrane inlet to a mass spectrometer. The depletion of /sup 18/O from CO/sub 2/ in the fluid outside the cells provides a method to study CO/sub 2/ and HCO/sub 3//sup -/ kinetics in suspensions of algae that contain carbonic anhydrase since /sup 18/O loss to H/sub 2/O is catalyzed inside the cells but not in the external fluid. Low-CO/sub 2/ cells of Chlorella vulgaris (grown with air) were added to a solution containing /sup 18/O enriched CO/sub 2/ and HCO/sub 3//sup -/ with 2 to 15 millimolar total inorganic carbon. The observed depletion of /sup 18/O from CO/sub 2/ was biphasic and the resulting /sup 18/O content of CO/sub 2/ was much less than the /sup 18/O content of HCO/sub 3//sup -/ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO/sub 3//sup -/ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 x 10/sup -4/ s/sup -1/ due to experimental errors. The Fick's law rate constant for entry of CO/sub 2/ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO/sub 2/ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO/sub 2/ entry into the cell or both. The CO/sub 2/ hydration activity inside the cells was 160 times the uncatalyzed CO/sub 2/ hydration rate.

  19. Novel alkalistable α-carbonic anhydrase from the polyextremophilic bacterium Bacillus halodurans: characteristics and applicability in flue gas CO2 sequestration.

    Science.gov (United States)

    Faridi, Shazia; Satyanarayana, T

    2016-08-01

    The emissions of CO2 into the atmosphere have been constantly rising due to anthropogenic activities, which have led to global warming and climate change. Among various methods proposed for mitigating CO2 levels in the atmosphere, carbonic anhydrase (CA)-mediated carbon sequestration represents a greener and safer approach to capture and convert it into stable mineral carbonates. Despite the fact that CA is an extremely efficient metalloenzyme that catalyzes the hydration of CO2 (CO2 + H2O ↔ HCO3 (-) + H(+)) with a kcat of ∼10(6) s(-1), a thermostable, and alkalistable CA is desirable for the process to take place efficiently. The purified CA from alkaliphilic, moderately thermophilic, and halotolerant Bacillus halodurans TSLV1 (BhCA) is a homodimeric enzyme with a subunit molecular mass of ~37 kDa with stability in a broad pH range between 6.0 and 11.0. It has a moderate thermostability with a T1/2 of 24.0 ± 1.0 min at 60 °C. Based on the sensitivity of CA to specific inhibitors, BhCA is an α-CA; this has been confirmed by nucleotide/amino acid sequence analysis. This has a unique property of stimulation by SO4 (2-), and it remains unaffected by SO3 (2-), NOx, and most other components present in the flue gas. BhCA is highly efficient in accelerating the mineralization of CO2 as compared to commercial bovine carbonic anhydrase (BCA) and is also efficient in the sequestration of CO2 from the exhaust of petrol driven car, thus, a useful biocatalyst for sequestering CO2 from flue gas. PMID:27102616

  20. Gene encoding γ-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7

    Directory of Open Access Journals (Sweden)

    Mishra Mukti N

    2010-07-01

    Full Text Available Abstract Background Carbonic anhydrase (CA is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of Methanosarcina thermophila (Cam has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one β-CA and two γ-CAs. Results One of the putative γ-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1. Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. Conclusions This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

  1. Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase I, II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines.

    Science.gov (United States)

    Abdel Gawad, Nagwa M; Amin, Noha H; Elsaadi, Mohammed T; Mohamed, Fatma M M; Angeli, Andrea; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T

    2016-07-01

    A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX were included in the study. The new sulfonamides showed excellent inhibition of all three isoforms, with KIs in the range of 0.84-702nM against hCA I, of 0.41-288nM against hCA II and of 5.6-29.2 against the tumor-associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new compounds showed micromolar inhibition of growth efficacy against breast cancer MCF-7 cell lines. PMID:27234893

  2. N-ethyl-N-nitrosourea-induced null mutation at the mouse Car-2 locus: An animal model for human carbonic anhydrase II deficiency syndrome

    International Nuclear Information System (INIS)

    Electrophoretic screening of (C57BL/6J x DBA/2J)F1 progeny of male mice treated with N-ethyl-N-nitrosourea revealed a mouse that lacked the paternal carbonic anhydrase II (Ca II). Breeding tests showed that this trait was heritable and due to a null mutation at the Car-2 locus on chromosome 3. Like humans with the same inherited enzyme defect, animals homozygous for the new null allele are runted and have renal tubular acidosis. However, the prominent osteopetrosis found in humans with CA II deficiency could be detected even in very old homozygous null mice. A molecular analysis of the deficient mice shows that the mutant gene is not deleted and is transcribed. The CA II protein, which is normally expressed in most tissues, could not be detected by immunodiffusion analysis in any tissues of the CA II-deficient mice, suggesting a nonsense or a missense mutation at the Car-2 locus

  3. Carborane-Based Carbonic Anhydrase Inhibitors: Insight into CAII/CAIX Specificity from a High-Resolution Crystal Structure, Modeling, and Quantum Chemical Calculations

    Directory of Open Access Journals (Sweden)

    Pavel Mader

    2014-01-01

    Full Text Available Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs. Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.

  4. Carborane-Based Carbonic Anhydrase Inhibitors: Insight into CAII/CAIX Specificity from a High-Resolution Crystal Structure, Modeling, and Quantum Chemical Calculations

    Science.gov (United States)

    Mader, Pavel; Pecina, Adam; Cígler, Petr; Lepšík, Martin; Šícha, Václav; Hobza, Pavel; Grüner, Bohumír; Fanfrlík, Jindřich; Brynda, Jiří; Řezáčová, Pavlína

    2014-01-01

    Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively. PMID:25309911

  5. The impact of Carbonic Anhydrase on the partitioning of leaf and soil CO18O and COS gas exchange across scales

    Science.gov (United States)

    Wingate, L.; Wehr, R. A.; Commane, R.; Ogee, J.; Sauze, J.; Jones, S.; Launois, T.; Wohl, S.; Whelan, M.; Meredith, L. K.; Genty, B.; Gimeno, T.; Kesselmeier, J.; Bosc, A.; Cuntz, M.; Munger, J. W.; Nelson, D. D.; Saleska, S. R.; Wofsy, S. C.; Zahniser, M. S.

    2015-12-01

    Photosynthesis (GPP), the largest CO2 flux to the land surface, is currently estimated with considerable uncertainty at between 100-175 Pg C yr-1. More robust estimates of global GPP could be obtained from the atmospheric budgets of other valuable tracers, such as carbonyl sulfide (COS) or the oxygen isotopic composition (δ18O) of atmospheric CO2. However, quantifying GPP using these tracers hinges on a better understanding of how soil micro-organisms modify the atmospheric concentrations of CO18O and COS at large scales. In particular, understanding better the role and activity of the enzyme Carbonic Anhydrase (CA) in soil micro-organisms is critical. We present novel datasets and model simulations demonstrating the progress in the collection of multi-tracer field datasets and how a new generation of multi-tracer land surface models can provide valuable constraints on photosynthesis and respiration across scales.

  6. Metalloprotein-inhibitor binding: human carbonic anhydrase II as a model for probing metal-ligand interactions in a metalloprotein active site.

    Science.gov (United States)

    Martin, David P; Hann, Zachary S; Cohen, Seth M

    2013-11-01

    An ever-increasing number of metalloproteins are being discovered that play essential roles in physiological processes. Inhibitors of these proteins have significant potential for the treatment of human disease, but clinical success of these compounds has been limited. Herein, zinc(II)-dependent metalloprotein inhibitors in clinical use are reviewed, and the potential for using novel metal-binding groups (MBGs) in the design of these inhibitors is discussed. By using human carbonic anhydrase II as a model system, the nuances of MBG-metal interactions in the context of a protein environment can be probed. Understanding how metal coordination influences inhibitor binding may help in the design of new therapeutics targeting metalloproteins.

  7. Carbonic Anhydrase IX is Not a Predictor of Outcomes in Non-Metastatic Clear Cell Renal Cell Carcinoma - A Digital Analysis of Tissue Microarray

    Directory of Open Access Journals (Sweden)

    Marcelo Zerati

    2013-07-01

    Full Text Available Introduction The knowledge about the molecular biology of clear cell renal cell carcinoma (ccRCC is evolving, and Carbonic Anhydrase type IX (CA-IX has emerged as a potential prognostic marker in this challenging disease. However, most of the literature about CA-IX on ccRCC comes from series on metastatic cancer, with a lack of series on non-metastatic cancer. The objective is to evaluate the expression of CA-IX in a cohort of non-metastatic ccRCC, correlating with 1 overall survival, and 2 with established prognostic parameters (T stage, tumor size, Fuhrman nuclear grade, microvascular invasion and peri-renal fat invasion. Materials and Methods This is a retrospective cohort study. We evaluated 95 patients with non-metastatic clear cell renal cell carcinoma, as to the expression of CA-IX. The analyzed parameters where: overall survival (OS, TNM stage, tumor size (TS, Fuhrman nuclear grade (FNG, microvascular invasion (MVI, peri-renal fat invasion (PFI. We utilized a custom built tissue microarray, and the immunoexpression was digitally quantified using the Photoshop® software. Results: Th e mean follow-up time was 7.9 years (range 1.9 to 19.5 years. The analysis of CA-IX expression against the selected prognostic parameters showed no correlation. The results are as follows: Overall survival (p = 0.790; T stage (p = 0.179; tumor size (p = 0.143; grouped Fuhrman nuclear grade (p = 0.598; microvascular invasion (p = 0.685, and peri-renal fat invasion (p = 0.104. Conclusion Carbonic anhydrase type IX expression does not correlate with overall survival and conventional prognostic parameters in non-metastatic clear cell renal cell carcinoma.

  8. Production, purification, and characterization of a fusion protein of carbonic anhydrase from Neisseria gonorrhoeae and cellulose binding domain from Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhu [Univ. of Pittsburgh, PA (United States); Bartlow, Patrick [Univ. of Pittsburgh, PA (United States); Dilmore, Robert M. [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States); Soong, Yee [National Energy Technology Lab. (NETL), Pittsburgh, PA, (United States); Pan, Zhiwei [Univ. of Pittsburgh, PA (United States); Koepsel, Richard [Univ. of Pittsburgh, PA (United States); Ataai, Mohammad [Univ. of Pittsburgh, PA (United States)

    2009-01-01

    Carbon dioxide capture technologies have the potential to become an important climate change mitigation option through sequestration of gaseous CO2, A new concept for CO2 capture involves use of immobilized carbonic anhydrase (CA) that catalyzes the reversible hydration of CO2 to HCO3- and H+. Cost-efficient production of the enzyme and an inexpensive immobilization system are critical for development of economically feasible CA-based CO2 capture processes. An artificial, bifunctional enzyme containing CA from Neisseria gonorrhoeae and a cellulose binding domain (CBD) from Clostridium thermocellum was constructed with a His6 tag. The chimeric enzyme exhibited both CA activity and CBD binding affinity. This fusion enzyme is of particular interest due to its binding affinity for cellulose and retained CA activity, which could serve as the basis for improved technology to capture CO2 from flue gasses.

  9. The Role of Hypoxia-Inducible Factor-1α, Glucose Transporter-1, (GLUT-1 and Carbon Anhydrase IX in Endometrial Cancer Patients

    Directory of Open Access Journals (Sweden)

    Pawel Sadlecki

    2014-01-01

    Full Text Available Hypoxia-inducible factor-1α (HIF-1α, glucose transporter-1 (GLUT-1, and carbon anhydrase IX (CAIX are important molecules that allow adaptation to hypoxic environments. The aim of our study was to investigate the correlation between HIF-1α, GLUT-1, and CAIX protein level with the clinicopathological features of endometrial cancer patients. Materials and Methods. 92 endometrial cancer patients, aged 37–84, were enrolled to our study. In all patients clinical stage, histologic grade, myometrial invasion, lymph node, and distant metastases were determined. Moreover, the survival time was assessed. Immunohistochemical analyses were performed on archive formalin fixed paraffin embedded tissue sections. Results. High significant differences (P=0.0115 were reported between HIF-1α expression and the histologic subtype of cancer. Higher HIF-1α expression was associated with the higher risk of recurrence (P=0.0434. The results of GLUT-1 and CAIX expression did not reveal any significant differences between the proteins expression in the primary tumor and the clinicopathological features. Conclusion. The important role of HIF-1α in the group of patients with the high risk of recurrence and the negative histologic subtype of the tumor suggest that the expression of this factor might be useful in the panel of accessory pathomorphological tests and could be helpful in establishing more accurate prognosis in endometrial cancer patients.

  10. Genomic organization of the human gene (CA5) and pseudogene for mitochondrial carbonic anhydrase V and their localization to chromosomes 16q and 16p

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yoshiro; Sly, W.S.; Batanian, J.R. [St. Louis Univ. School of Medicine, MO (United States)] [and others

    1995-08-10

    Carbonic anhydrase V (CA V) is expressed in mitochondrial matrix in liver and several other tissues. It is of interest for its putative roles in providing bicarbonate to carbamoyl phosphate synthetase for ureagenesis and to pyruvate carboxylase for gluconeogenesis and its possible importance in explaining certain inherited metabolic disorders with hyperammonemia and hypoglycemia. Following the recent characterization of the cDNA for human CA V, we report the isolation of the human gene from two {lambda} genomic libraries and its characterization. The CA V gene (CA5) is approximately 50 kb long and contains 7 exons and 6 introns. The exon-intron boundaries are found in positions identical to those determined for the previously described CA II, CA III, and CA VII genes. Like the CA VII gene, CA5 does not contain typical TATA and CAAT promoter elements in the 5{prime} flanking region but does contain a TTTAA sequence 147 nucleotides upstream of the initiation codon. CA5 also contains a 12-bp GT-rich segment beginning 13 bp downstream of the polyadenylation signal in the 3{prime} untranslated region of exon 7. FISH analysis allowed CA5 to be assigned to chromosome 16q24.3. An unprocessed pseudogene containing sequence homologous to exons 3-7 and introns 3-6 was also isolated and was assigned by FISH analysis to chromosome 16p11.2-p12. 22 refs., 4 figs., 1 tab.

  11. Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma.

    Science.gov (United States)

    Alafeefy, Ahmed M; Carta, Fabrizio; Ceruso, Mariangela; Al-Tamimi, Abdul-Malek S; Al-Kahtani, Abdulla A; Supuran, Claudiu T

    2016-03-15

    A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (KIs of 81.0-3084 nM), being highly effective as hCA II (KIs in the range of 0.25-10.8 nM), IX (KIs of 3.7-50.4 nM) and XII (KIs of 0.60-52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated. PMID:26875933

  12. Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low nanomolar/subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform XII.

    Science.gov (United States)

    Eldehna, Wagdy M; Fares, Mohamed; Ceruso, Mariangela; Ghabbour, Hazem A; Abou-Seri, Sahar M; Abdel-Aziz, Hatem A; Abou El Ella, Dalal A; Supuran, Claudiu T

    2016-03-01

    By using a molecular hybridization approach, two series of amido/ureidosubstituted benzenesulfonamides incorporating substituted-isatin moieties were synthesized. The prepared derivatives were in vitro evaluated for their inhibitory activity against human carbonic anhydrase (hCA, EC 4.2.1.1) I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms. All these isoforms were inhibited in variable degrees by the sulfonamides reported here. hCA I was inhibited with KIs in the range of 7.9-894 nM, hCA II in the range of 7.5-1645 nM (with one compound having a KI > 10 μM); hCA IX in the range of 5.0-240 nM, whereas hCA XII in the range of 0.47-2.83 nM. As all these isoforms are involved in various pathologies, in which their inhibition can be exploited therapeutically, the derivatives reported here may represent interesting extensions to the field of CA inhibitors of the sulfonamide type. PMID:26840366

  13. The impact of hydroquinone on acetylcholine esterase and certain human carbonic anhydrase isoenzymes (hCA I, II, IX, and XII).

    Science.gov (United States)

    Scozzafava, Andrea; Kalın, Pınar; Supuran, Claudiu T; Gülçin, İlhami; Alwasel, Saleh H

    2015-12-01

    Carbonic anhydrases (CAs) are widespread and the most studied members of a great family of metalloenzymes in higher vertebrates including humans. CAs were investigated for their inhibition of all of the catalytically active mammalian isozymes of the Zn(2+)-containing CA, (CA, EC 4.2.1.1). On the other hand, acetylcholinesterase (AChE. EC 3.1.1.7), a serine protease, is responsible for ACh hydrolysis and plays a fundamental role in impulse transmission by terminating the action of the neurotransmitter ACh at the cholinergic synapses and neuromuscular junction. In the present study, the inhibition effect of the hydroquinone (benzene-1,4-diol) on AChE activity was evaluated and effectively inhibited AChE with Ki of 1.22 nM. Also, hydroquinone strongly inhibited some human cytosolic CA isoenzymes (hCA I and II) and tumour-associated transmembrane isoforms (hCA IX, and XII), with Kis in the range between micromolar (415.81 μM) and nanomolar (706.79 nM). The best inhibition was observed in cytosolic CA II. PMID:25586344

  14. Microwave assisted synthesis of novel acridine-acetazolamide conjugates and investigation of their inhibition effects on human carbonic anhydrase isoforms hCA I, II, IV and VII.

    Science.gov (United States)

    Ulus, Ramazan; Aday, Burak; Tanç, Muhammet; Supuran, Claudiu T; Kaya, Muharrem

    2016-08-15

    4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide was condensed with cyclic-1,3-diketones (dimedone and cyclohexane-1,3-dione) and aromatic aldehydes under microwave irradiation, leading to a series of acridine-acetazolamide conjugates. The new compounds were investigated as inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), and more precisely cytosolic isoforms hCA I, II, VII and membrane-bound one hCA IV. All investigated isoforms were inhibited in low micromolar and nanomolar range by the new compounds. hCA IV and VII were inhibited with KIs in the range of 29.7-708.8nM (hCA IV), and of 1.3-90.7nM (hCA VII). For hCA I and II the KIs were in the range of 6.7-335.2nM (hCA I) and of 0.5-55.4nM (hCA II). The structure-activity relationships (SAR) for the inhibition of these isoforms with the acridine-acetazolamide conjugates reported here were delineated. PMID:27298005

  15. Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties.

    Science.gov (United States)

    Bozdag, Murat; Alafeefy, Ahmed M; Carta, Fabrizio; Ceruso, Mariangela; Al-Tamimi, Abdul-Malek S; Al-Kahtani, Abdulla A; Alasmary, Fatmah A S; Supuran, Claudiu T

    2016-09-15

    Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (KIs of 28.5-2954nM), being highly effective as hCA II (KIs in the range of 0.62-12.4nM) and XII (KIs of 0.54-7.11nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated. PMID:27396930

  16. Oxygen-18 isotope of breath CO₂ linking to erythrocytes carbonic anhydrase activity: a biomarker for pre-diabetes and type 2 diabetes.

    Science.gov (United States)

    Ghosh, Chiranjit; Banik, Gourab D; Maity, Abhijit; Som, Suman; Chakraborty, Arpita; Selvan, Chitra; Ghosh, Shibendu; Chowdhury, Subhankar; Pradhan, Manik

    2015-01-01

    Carbonic anhydrase (CA), a well-characterized metalloenzyme, is associated with oxygen-18 ( (18)O)-isotopic fractionations of CO₂. To investigate how CA activity links the (18)O of breath CO₂ to pre-diabetes (PD) and type 2 diabetes (T2D) during metabolism, we studied pre- and post-dose CA activities in erythrocytes with simultaneous monitoring of (18)O/ (16)O-isotope ratios of breath CO₂ and thereafter elucidated potential metabolic pathways underlying CA alteration in the pathogenesis of T2D. Here we show that the post-dose CA activity in both T2D and PD was markedly enhanced, whereas the non-diabetic controls (NDC) exhibited a considerable reduction in post-dose CA activity when compared with their basal CA activities. However, T2D and PD exhibited isotopic enrichments of (18)O in breath CO₂, while a marked depletion of (18)O in CO₂ was manifested in NDC. Thus, the isotopic enrichments and depletions of (18)O in breath CO₂ were well correlated with the changes in CA activities for controls, PD and T2D. Our findings suggest the changes in CA activities in erythrocytes may contribute to the pathogenesis of T2D and the breath C (18)O (16)O regulated by the CA activity as a potential biomarker for non-invasive assessment of T2D, and thus may open a new method for treating T2D. PMID:25633556

  17. Oxygen-18 isotope of breath CO2 linking to erythrocytes carbonic anhydrase activity: a biomarker for pre-diabetes and type 2 diabetes

    Science.gov (United States)

    Ghosh, Chiranjit; Banik, Gourab D.; Maity, Abhijit; Som, Suman; Chakraborty, Arpita; Selvan, Chitra; Ghosh, Shibendu; Chowdhury, Subhankar; Pradhan, Manik

    2015-01-01

    Carbonic anhydrase (CA), a well-characterized metalloenzyme, is associated with oxygen-18 ( 18O)-isotopic fractionations of CO2. To investigate how CA activity links the 18O of breath CO2 to pre-diabetes (PD) and type 2 diabetes (T2D) during metabolism, we studied pre- and post-dose CA activities in erythrocytes with simultaneous monitoring of 18O/ 16O-isotope ratios of breath CO2 and thereafter elucidated potential metabolic pathways underlying CA alteration in the pathogenesis of T2D. Here we show that the post-dose CA activity in both T2D and PD was markedly enhanced, whereas the non-diabetic controls (NDC) exhibited a considerable reduction in post-dose CA activity when compared with their basal CA activities. However, T2D and PD exhibited isotopic enrichments of 18O in breath CO2, while a marked depletion of 18O in CO2 was manifested in NDC. Thus, the isotopic enrichments and depletions of 18O in breath CO2 were well correlated with the changes in CA activities for controls, PD and T2D. Our findings suggest the changes in CA activities in erythrocytes may contribute to the pathogenesis of T2D and the breath C 18O 16O regulated by the CA activity as a potential biomarker for non-invasive assessment of T2D, and thus may open a new method for treating T2D. PMID:25633556

  18. Effect of CO2 concentrations on the activity of photosynthetic CO2 fixation and extracelluar carbonic anhydrase in the marine diatom Skeletonema costatum

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiongwen; GAO Kunshan

    2003-01-01

    The growth and activity of photosynthetic CO2 uptake and extracellular carbonic anhydrase (Caext) of the marine diatom Skeletonema costatum were investigated while cultured at different levels of CO2 in order to see its physiological response to different CO2 concentrations under either a low (30 μmol·m-2·s-1) or high (210 μmol·m-2·s-1) irradiance. The changes in CO2 concentrations (4-31 μmol/L) affected the growth and net photosynthesis to a greater extent under the low than under the high light regime. Caext was detected in the cells grown at 4 μmol/L CO2 but not at 31 and 12 μmol/L CO2, with its activity being about 2.5-fold higher at the high than at the low irradiance. Photo- synthetic CO2 affinity (1/ K1/2(CO2)) of the cells decreased with increased CO2 concentrations in culture. The cells cultured under the high-light show significantly higher photosynthetic CO2 affinity than those grown at the low-light level. It is concluded that the regulations of Caext activity and photosynthetic CO2 affinity are dependent not only on CO2 concentration but also on light availability, and that the development of higher Caext activity and CO2 affinity under higher light level could sufficiently support the photosynthetic demand for CO2 even at low level of CO2.

  19. A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.

    Science.gov (United States)

    Pinard, Melissa A; Aggarwal, Mayank; Mahon, Brian P; Tu, Chingkuang; McKenna, Robert

    2015-10-01

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

  20. Expression and characterization of a recombinant psychrophilic γ-carbonic anhydrase (NcoCA) identified in the genome of the Antarctic cyanobacteria belonging to the genus Nostoc.

    Science.gov (United States)

    De Luca, Viviana; Del Prete, Sonia; Vullo, Daniela; Carginale, Vincenzo; Di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-10-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO2 hydration/dehydration reversible reaction: CO2 + H2O ⇄ [Formula: see text] + H(+). Living organisms encode for at least six distinct genetic families of such catalyst, the α-, β-, γ-, δ-, ζ- and η-CAs. The main function of the CAs is to quickly process the CO2 derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H(+)) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average -1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a γ-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to α-, β-, γ-classes, as well as a limited stability at moderate temperatures. PMID:26226178

  1. Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity.

    Science.gov (United States)

    Kucuk, Murat; Gulcin, İlhami

    2016-06-01

    In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77EU/mg and a yield of 35.5% using Sepharose-4B-l-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71kDa. The optimum temperature, activation energy (Ea), activation enthalpy (ΔH) and Q10 values were obtained from Arrhenius plot. Km and Vmax values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe(2+), Pb(2+), Co(2+), Ag(+) and Cu(2+)) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC50) were obtained. Finally Ki values and inhibition types were calculated from Lineweaver-Burk graphs. PMID:27175889

  2. Targeting carbonic anhydrase IX by nitroimidazole based sulfamides enhances the therapeutic effect of tumor irradiation: A new concept of dual targeting drugs

    International Nuclear Information System (INIS)

    Background and purpose: Carbonic anhydrase IX (CAIX) plays an important role in pH regulation processes critical for tumor cell growth and metastasis. We hypothesize that a dual targeting bioreductive nitroimidazole based anti-CAIX sulfamide drug (DH348) will reduce tumor growth and sensitize tumors to irradiation in a CAIX dependent manner. Material and methods: The effect of the dual targeting anti-CAIX (DH348) and its single targeting control drugs on extracellular acidification and radiosensitivity was examined in HT-29 colorectal carcinoma cells. Tumor growth and time to reach 4× start volume (T4×SV) was monitored for animals receiving DH348 (10 mg/kg) combined with tumor single dose irradiation (10 Gy). Results: In vitro, DH348 reduced hypoxia-induced extracellular acidosis, but did not change hypoxic radiosensitivity. In vivo, DH348 monotherapy decreased tumor growth rate and sensitized tumors to radiation (enhancement ratio 1.50) without systemic toxicity only for CAIX expressing tumors. Conclusions: A newly designed nitroimidazole and sulfamide dual targeting drug reduces hypoxic extracellular acidification, slows down tumor growth at nontoxic doses and sensitizes tumors to irradiation all in a CAIX dependent manner, suggesting no “off-target” effects. Our data therefore indicate the potential utility of a dual drug approach as a new strategy for tumor-specific targeting

  3. Association between dental-oral health in young adults and salivary glutathione, lipid peroxidation and sialic acid levels and carbonic anhydrase activity

    Directory of Open Access Journals (Sweden)

    L.K. Öztürk

    2008-11-01

    Full Text Available The aim of the present study was to evaluate the relationship between salivary oxidative stress and dental-oral health. Healthy young adults, matched for gender and age, with (N = 21, 10 men, mean age: 20.3 ± 1 years and without (N = 16, 8 men, mean age: 21.2 ± 1.8 years caries were included in this study. The World Health Organization (WHO caries diagnostic criteria were used for determining the decayed, missing, filled teeth (DMFT index. The oral hygiene and gingival status were assessed using the simplified oral hygiene index and gingival index, respectively. Unstimulated salivary total protein, glutathione (GSH, lipid peroxidation and total sialic acid levels, carbonic anhydrase activity, and salivary buffering capacity were determined by standard methods. Furthermore, salivary pH was measured with pH paper and salivary flow rate was calculated. Simplified oral hygiene index and gingival index were not significantly different between groups but DMFT scores were significant (P < 0.01. Only, GSH values were significantly different (P < 0.05 between groups (2.2 and 1.6 mg/g protein in young adults without caries and with caries, respectively. There was a significant negative correlation between DMFT and GSH (r = -0.391; P < 0.05; Pearson's correlation coefficient. Our results suggest that there is an association between caries history and salivary GSH levels.

  4. Expression and characterization of a recombinant psychrophilic γ-carbonic anhydrase (NcoCA) identified in the genome of the Antarctic cyanobacteria belonging to the genus Nostoc.

    Science.gov (United States)

    De Luca, Viviana; Del Prete, Sonia; Vullo, Daniela; Carginale, Vincenzo; Di Fonzo, Pietro; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-10-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO2 hydration/dehydration reversible reaction: CO2 + H2O ⇄ [Formula: see text] + H(+). Living organisms encode for at least six distinct genetic families of such catalyst, the α-, β-, γ-, δ-, ζ- and η-CAs. The main function of the CAs is to quickly process the CO2 derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H(+)) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average -1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a γ-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to α-, β-, γ-classes, as well as a limited stability at moderate temperatures.

  5. Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I, II, VII, XII, and XIII.

    Directory of Open Access Journals (Sweden)

    Miglė Kišonaitė

    Full Text Available The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthioacetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.

  6. The most recently discovered carbonic anhydrase, CA XV, is expressed in the thick ascending limb of Henle and in the collecting ducts of mouse kidney.

    Directory of Open Access Journals (Sweden)

    Sina Saari

    Full Text Available BACKGROUND: Carbonic anhydrases (CAs are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla. CONCLUSION/SIGNIFICANCE: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis.

  7. Carbonic anhydrase activity and photosynthetic rate in the tree species Paulownia tomentosa Steud. Effect of dimethylsulfoxide treatment and zinc accumulation in leaves.

    Science.gov (United States)

    Lazova, Galia N; Naidenova, Tsveta; Velinova, Katya

    2004-03-01

    The enzyme carbonic anhydrase (CA) (EC 4.2.1.1) catalyzes the reversible conversion of CO2 to HCO3- and has been shown to be involved in photosynthesis. The enzyme has been shown in animals, plants, eubacteria and viruses, but similar reports on the evidence for CA activity in tree plants does not be appear to be available. In the preliminary analyses of the work, the CA activity in leaf extracts from the tree species Paulownia tomentosa Steud. (introduced in Bulgaria) is described. A connection between CA activity and the rate of photosynthetic CO2 fixation is shown. In the second portion of the work, the effect of 10(-4) mol/L and 10(-2) mol/L dimethylsulfoxide (DMSO) on the zinc accumulation in leaves is demonstrated. It is suggested that CA activity is an indicator of the level of physiologically active zinc in leaves of P. tomentosa Steud. A connection between the process of zinc accumulation in leaves and the activity of the enzymes CA and glycolate oxidase (GO) (EC 1.1.3.1) is established. PMID:15077628

  8. Anion inhibition studies of the α-carbonic anhydrase from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease.

    Science.gov (United States)

    Pan, Peiwen; Vermelho, Alane Beatriz; Scozzafava, Andrea; Parkkila, Seppo; Capasso, Clemente; Supuran, Claudiu T

    2013-08-01

    The protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease, encodes an α-class carbonic anhydrase (CA, EC 4.2.1.1), TcCA, which was recently shown to be crucial for its life cycle. Thiols, a class of strong TcCA inhibitors, were also shown to block the growth of the pathogen in vitro. Here we report the inhibition of TcCA by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, phenylboronic/arsonic acids. TcCA was inhibited in the low micromolar range by iodide, cyanate, thiocyanate, hydrogensulfide and trithiocarbonate (KIs in the range of 44-93 μM), but the best inhibitor was diethyldithiocarbamate (KI=5 μM). Sulfamide showed an inhibition constant of 120 μM, but sulfamic acid was much less effective (KI of 10.6 mM). The discovery of diethyldithiocarbamate as a low micromolar TcCA inhibitor may be useful to detect leads for developing anti-Trypanosoma agents with a diverse mechanism of action compared to clinically used drugs (benznidazole, nifurtimox) for which significant resistance emerged. PMID:23790722

  9. The selective expression of carbonic anhydrase genes of Aspergillus nidulans in response to changes in mineral nutrition and CO2 concentration.

    Science.gov (United States)

    Xiao, Leilei; Lian, Bin; Dong, Cuiling; Liu, Fanghua

    2016-02-01

    Carbonic anhydrase (CA) plays an important role in the formation and evolution of life. However, to our knowledge, there has been no report on CA isoenzyme function differentiation in fungi. Two different CA gene sequences in Aspergillus nidulans with clear genetic background provide us a favorable basis for studying function differentiation of CA isoenzymes. Heterologously expressed CA1 was used to test its weathering ability on silicate minerals and real-time quantitative PCR was used to detect expression of the CA1 and CA2 genes at different CO2 concentrations and in the presence of different potassium sources. The northern blot method was applied to confirm the result of CA1 gene expression. Heterologously expressed CA1 significantly promoted dissolution of biotite and wollastonite, and CA1 gene expression increased significantly in response to soluble K-deficiency. The northern blot test further showed that CA1 participated in K-feldspar weathering. In addition, the results showed that CA2 was primary involved in adapting to CO2 concentration change. Taken together, A. nidulans can choose different CA to meet their survival needs, which imply that some environmental microbes have evolved different CAs to adapt to changes in CO2 concentration and acquire mineral nutrition so that they can better adapt to environmental changes. Inversely, their adaption may impact mineral weathering and/or CO2 concentration, and even global change.

  10. In Vivo Loss of Function Screening Reveals Carbonic Anhydrase IX as a Key Modulator of Tumor Initiating Potential in Primary Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Nabendu Pore

    2015-06-01

    Full Text Available Reprogramming of energy metabolism is one of the emerging hallmarks of cancer. Up-regulation of energy metabolism pathways fuels cell growth and division, a key characteristic of neoplastic disease, and can lead to dependency on specific metabolic pathways. Thus, targeting energy metabolism pathways might offer the opportunity for novel therapeutics. Here, we describe the application of a novel in vivo screening approach for the identification of genes involved in cancer metabolism using a patient-derived pancreatic xenograft model. Lentiviruses expressing short hairpin RNAs (shRNAs targeting 12 different cell surface protein transporters were separately transduced into the primary pancreatic tumor cells. Transduced cells were pooled and implanted into mice. Tumors were harvested at different times, and the frequency of each shRNA was determined as a measure of which ones prevented tumor growth. Several targets including carbonic anhydrase IX (CAIX, monocarboxylate transporter 4, and anionic amino acid transporter light chain, xc- system (xCT were identified in these studies and shown to be required for tumor initiation and growth. Interestingly, CAIX was overexpressed in the tumor initiating cell population. CAIX expression alone correlated with a highly tumorigenic subpopulation of cells. Furthermore, CAIX expression was essential for tumor initiation because shRNA knockdown eliminated the ability of cells to grow in vivo. To the best of our knowledge, this is the first parallel in vivo assessment of multiple novel oncology target genes using a patient-derived pancreatic tumor model.

  11. The alpha-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 is highly susceptible to inhibition by sulfonamides.

    Science.gov (United States)

    Vullo, Daniela; Luca, Viviana De; Scozzafava, Andrea; Carginale, Vincenzo; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2013-03-15

    The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its inhibition with a large series of sulfonamides and a sulfamate, the classical inhibitors of these zinc enzymes. SspCA showed an inhibition profile with these compounds very similar to that of the predominant human cytosolic isoform hCA II, and not to that of the bacterial α-CA from Helicobacter pylori. Some clinically used drugs such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, celecoxib and sulthiame were low nanomolar SspCA/hCA II inhibitors (KIs in the range of 4.5-12.3nM) whereas simple aromatic/heterocyclic sulfonamides were less effective, micromolar inhibitors. As this highly catalytically active and thermostable enzyme may show biotechnological applications, its inhibition studies may be relevant for designing on/off systems to control its activity. PMID:22883029

  12. Synthesis and In Vitro Inhibition Effect of New Pyrido[2,3-d]pyrimidine Derivatives on Erythrocyte Carbonic Anhydrase I and II

    Directory of Open Access Journals (Sweden)

    Hilal Kuday

    2014-01-01

    Full Text Available In vitro inhibition effects of indolylchalcones and new pyrido[2,3-d]pyrimidine derivatives on purified human carbonic anhydrase I and II (hCA I and II were investigated by using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among all the synthesized compounds, 7e (IC50=6.79 µM was found to be the most active compound for hCA I inhibitory activity and 5g (IC50=7.22 µM showed the highest hCA II inhibitory activity. Structure-activity relationships study showed that indolylchalcone derivatives have higher inhibitory activities than pyrido[2,3-d]pyrimidine derivatives on hCA I and hCA II. Additionally, methyl group bonded to uracil ring increases inhibitory activities on both hCA I and hCA II.

  13. Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate.

    Science.gov (United States)

    Del Prete, Sonia; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T; Carginale, Vincenzo

    2016-01-15

    With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.

  14. Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate.

    Science.gov (United States)

    Del Prete, Sonia; De Luca, Viviana; Capasso, Clemente; Supuran, Claudiu T; Carginale, Vincenzo

    2016-01-15

    With the continuous increase of atmospheric CO2 in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO2 into useful chemicals could be a good strategy to both decreasing the CO2 concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO2 mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO2 to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO2 into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO2 into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO2 hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes. PMID:26712095

  15. Comparison of the sulfonamide inhibition profiles of the α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae.

    Science.gov (United States)

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Carginale, Vincenzo; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T; Capasso, Clemente

    2016-04-15

    Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the conversion of carbon dioxide (CO2) to bicarbonate (HCO3(-)) and protons (H(+)). In prokaryotes, the existence of genes encoding for α-, β- and γ-classes suggests that these enzymes play an important role in the prokaryotic physiology. It has been demonstrated, in fact, that their inhibition in vivo leads to growth impairment or growth defects of the microorganism. Ultimately, we started to investigate the biochemical properties and the inhibitory profiles of the α- and β-CAs identified in the genome of Vibrio cholerae, which is the causative agent of cholera. The genome of this pathogen encodes for CAs belonging to α, β and γ classes. Here, we report a sulfonamide inhibition study of the γ-CA (named VchCAγ) comparing it with data obtained for the α- and β-CA enzymes. VchCAγ activity (kcat=7.39 × 10(5)s(-1)) was significantly higher than the other γ-CAs. The inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of nanomolar VchCAγ inhibitors, including simple aromatic/heterocyclic sulfonamides (compounds 2-9, 11, 13-15, 24) as well as EZA, DZA, BRZ, BZA, TPM, ZNS, SLP, IND (KIs in the range of 66.2-95.3 nM). As it was proven that bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit this virulence in vivo, we propose that VchCA, VchCAβ and VchCAγ may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now, i.e., interference with bicarbonate supply as a virulence factor. PMID:26972117

  16. Roles of Carbonic Anhydrase in Photosynthesis of Skeletonema costatum%碳酸酐酶在中肋骨条藻光合作用中的作用

    Institute of Scientific and Technical Information of China (English)

    陈雄文; 高坤山

    2004-01-01

    探讨了在正常空气条件下生长的中肋骨条藻(Skeletonema costatum)的碳酸酐酶(CA)在其光合固碳中的作用.在中肋骨条藻的胞内和胞外均有CA活性,但胞外CA活性很低.CA抑制剂AZ(乙酰唑磺胺)对中肋骨条藻的光合放氧速率没有明显影响,而CA抑制剂EZ(乙氧苯唑胺)对其光合放氧速率有强烈的抑制作用.EZ的抑制作用使细胞最大光合速率、饱和光强和无机碳亲和力下降,无机碳的补偿点和光呼吸提高,使强光下光抑制作用增强.这些结果表明:中肋骨条藻的胞外CA在其光合作用中所起的作用较小,而其胞内CA通过催化胞内碳库中的HCO-3快速转化成CO2,提高胞内CO2的有效供给,从而提高细胞光合固碳能力和对逆境(高O2、强光和低CO2)的适应能力.%The role of carbonic anhydrase (CA) in photosynthesis of the marine diatom Skeletonema costatum grown at ambient level of CO2 was investigated. Extracellular CA activity was very low. In comparison, intracellular CA activity was great part of total CA activity. The inhibition of external CA by acetazolamide (AZ) caused little change in net photosynthetic rate (Pn), but the inhibition of intracellular CA by ethoxyzolamide (EZ) resulted in the strong reduction of Pn. EZ reduced the light-saturated photosynthesis, the saturation radiance and the affin ity of inorganic carbon for photosynthesis, raised inorganic carbon compensation point and enhanced the inhibition of photosynthesis by high O2 and light. It is concluded that extracellular CA exerted a minor role in the photosynthesis, but intracellular CA enhanced the efficiency of photosynthetic carbon fixation and the capacity of acclimation to stress conditions (high light, O2 and low CO2) by catalytically converting HCO-3 to CO2 and facilitating CO2 supply to the cell.

  17. Label-free characterization of carbonic anhydrase-novel inhibitor interactions using surface plasmon resonance, isothermal titration calorimetry and fluorescence-based thermal shift assays.

    Science.gov (United States)

    Rogez-Florent, Tiphaine; Duhamel, Laetitia; Goossens, Laurence; Six, Perrine; Drucbert, Anne-Sophie; Depreux, Patrick; Danzé, Pierre-Marie; Landy, David; Goossens, Jean-François; Foulon, Catherine

    2014-01-01

    This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka , kd , and KD ), leads us to choose this methodology for the study of new potential inhibitors. PMID:24375583

  18. Kinetic and X-ray crystallographic investigations on carbonic anhydrase isoforms I, II, IX and XII of a thioureido analog of SLC-0111.

    Science.gov (United States)

    Lomelino, Carrie L; Mahon, Brian P; McKenna, Robert; Carta, Fabrizio; Supuran, Claudiu T

    2016-03-01

    SLC-0111 (4-(4-fluorophenylureido)-benzenesulfonamide) is the first carbonic anhydrase (CA, EC 4.2.1.1) IX inhibitor to reach phase I clinical trials as an antitumor/antimetastatic agent. Here we report a kinetic and X-ray crystallographic study of a congener of SLC-0111 which incorporates a thioureido instead of ureido linker between the two aromatic rings as inhibitor of four physiologically relevant CA isoforms. Similar to SLC-0111, the thioureido derivative was a weak hCA I and II inhibitor and a potent one against hCA IX and XII. X-ray crystallography of its adduct with hCA II and comparison of the structure with that of other five hCA II-sulfonamide adducts belonging to the SLC-0111 series, afforded us to understand the particular inhibition profile of the new sulfonamide. Similar to SLC-0111, the thioureido sulfonamide primarily interacted with the hydrophobic side of the hCA II active site, with the tail participating in van der Waals interactions with Phe131 and Pro202, in addition to the coordination of the deprotonated sulfonamide to the active site metal ion. On the contrary, the tail of other sulfonamides belonging to the SLC-0111 series (2-isopropyl-phenyl; 3-nitrophenyl) were orientated towards the hydrophilic half of the active site, which was correlated with orders of magnitude better inhibitory activity against hCA II, and a loss of selectivity for the inhibition of the tumor-associated CAs. PMID:26810836

  19. Neutron structure of human carbonic anhydrase II in complex with methazolamide: mapping the solvent and hydrogen-bonding patterns of an effective clinical drug

    Directory of Open Access Journals (Sweden)

    Mayank Aggarwal

    2016-09-01

    Full Text Available Carbonic anhydrases (CAs; EC 4.2.1.1 catalyze the interconversion of CO2 and HCO3−, and their inhibitors have long been used as diuretics and as a therapeutic treatment for many disorders such as glaucoma and epilepsy. Acetazolamide (AZM and methazolamide (MZM, a methyl derivative of AZM are two of the classical CA inhibitory drugs that have been used clinically for decades. The jointly refined X-ray/neutron structure of MZM in complex with human CA isoform II (hCA II has been determined to a resolution of 2.2 Å with an Rcryst of ∼16.0%. Presented in this article, along with only the second neutron structure of a clinical drug-bound hCA, is an in-depth structural comparison and analyses of differences in hydrogen-bonding network, water-molecule orientation and solvent displacement that take place upon the binding of AZM and MZM in the active site of hCA II. Even though MZM is slightly more hydrophobic and displaces more waters than AZM, the overall binding affinity (Ki for both of the drugs against hCA II is similar (∼10 nM. The plausible reasons behind this finding have also been discussed using molecular dynamics and X-ray crystal structures of hCA II–MZM determined at cryotemperature and room temperature. This study not only allows a direct comparison of the hydrogen bonding, protonation states and solvent orientation/displacement of AZM and MZM, but also shows the significant effect that the methyl derivative has on the solvent organization in the hCA II active site.

  20. High kinetic stability of Zn(II) coordinated by the tris(histidine) unit of carbonic anhydrase towards solvolytic dissociation studied by affinity capillary electrophoresis.

    Science.gov (United States)

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko

    2016-08-01

    Solvolytic dissociation rate constants (kd) of bovine carbonic anhydrase II (CA) and its metallovariants (M-CAs, M=Co(II), Ni(II), Cu(II), Zn(II), and Cd(II)) were estimated by a ligand substitution reaction, which was monitored by affinity capillary electrophoresis to selectively detect the undissociated CAs in the reaction mixture. Using EDTA as the competing ligand for Zn-CA, the dissociation followed the unimolecular nucleophilic substitution (SN1) mechanism with kd=1.0×10(-7)s(-1) (pH7.4, 25°C). The corresponding solvolysis half-life (t1/2) was 80days, showing the exceptionally high kinetic stability of t Zn-CA, in contrast to the highly labile [Zn(II)(H2O)6](2+), where the water exchange rate (kex) is high. This behavior is attributed to the tetrahedral coordination geometry supported by the tris(histidine) unit (His3) of CA. In the case of Co-CA, it showed a somewhat larger kd value (5.7×10(-7)s(-1), pH7.4, 25°C) even though it shares the same tetrahedral coordination environment with Zn-CA, suggesting that the d(7) electronic configuration of Co(II) in the transition state of the dissociation is stabilized by the ligand field. Among M-CAs, only Ni-CA showed a bimolecular nucleophilic substitution (SN2) reaction path in its reaction with EDTA, implying that the large coordination number (6) of Ni(II) in Ni-CA allows EDTA to form an EDTA-Ni-CA intermediate. Overall, kd values roughly correlated with kex values among M-CAs, with the kd value of Zn-CA deviating strongly from the trend and highlighting the exceptionally high kinetic stabilization of Zn-CA by the His3 unit. PMID:27235274

  1. The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932-2015).

    Science.gov (United States)

    Buzás, György M; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Puşcaş used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Puşcaş for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend. PMID:26108882

  2. Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor.

    Science.gov (United States)

    Wang, Cun; Hu, Honghong; Qin, Xue; Zeise, Brian; Xu, Danyun; Rappel, Wouter-Jan; Boron, Walter F; Schroeder, Julian I

    2016-02-01

    Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric [CO2] further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO2/HCO3 (-) enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels. PMID:26764375

  3. Label-free characterization of carbonic anhydrase-novel inhibitor interactions using surface plasmon resonance, isothermal titration calorimetry and fluorescence-based thermal shift assays.

    Science.gov (United States)

    Rogez-Florent, Tiphaine; Duhamel, Laetitia; Goossens, Laurence; Six, Perrine; Drucbert, Anne-Sophie; Depreux, Patrick; Danzé, Pierre-Marie; Landy, David; Goossens, Jean-François; Foulon, Catherine

    2014-01-01

    This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka , kd , and KD ), leads us to choose this methodology for the study of new potential inhibitors.

  4. Modelling the impact of soil Carbonic Anhydrase on the net ecosystem exchange of OCS at Harvard forest using the MuSICA model

    Science.gov (United States)

    Launois, Thomas; Ogée, Jérôme; Commane, Roisin; Wehr, Rchard; Meredith, Laura; Munger, Bill; Nelson, David; Saleska, Scott; Wofsy, Steve; Zahniser, Mark; Wingate, Lisa

    2016-04-01

    The exchange of CO2 between the terrestrial biosphere and the atmosphere is driven by photosynthetic uptake and respiratory loss, two fluxes currently estimated with considerable uncertainty at large scales. Model predictions indicate that these biosphere fluxes will be modified in the future as CO2 concentrations and temperatures increase; however, it still unclear to what extent. To address this challenge there is a need for better constraints on land surface model parameterisations. Additional atmospheric tracers of large-scale CO2 fluxes have been identified as potential candidates for this task. In particular carbonyl sulphide (OCS) has been proposed as a complementary tracer of gross photosynthesis over land, since OCS uptake by plants is dominated by carbonic anhydrase (CA) activity, an enzyme abundant in leaves that catalyses CO2 hydration during photosynthesis. However, although the mass budget at the ecosystem is dominated by the flux of OCS into leaves, some OCS is also exchanged between the atmosphere and the soil and this component of the budget requires constraining. In this study, we adapted the process-based isotope-enabled model MuSICA (Multi-layer Simulator of the Interactions between a vegetation Canopy and the Atmosphere) to include the transport, reaction, diffusion and production of OCS within a forested ecosystem. This model was combined with 3 years (2011-2013) of in situ measurements of OCS atmospheric concentration profiles and fluxes at the Harvard Forest (Massachussets, USA) to test hypotheses on the mechanisms responsible for CA-driven uptake by leaves and soils as well as possible OCS emissions during litter decomposition. Model simulations over the three years captured well the impact of diurnally and seasonally varying environmental conditions on the net ecosystem OCS flux. A sensitivity analysis on soil CA activity and soil OCS emission rates was also performed to quantify their impact on the vertical profiles of OCS inside the

  5. The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932-2015).

    Science.gov (United States)

    Buzás, György M; Supuran, Claudiu T

    2016-08-01

    Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Puşcaş used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Puşcaş for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend.

  6. Complexes With Biologically Active Ligands. Part 111. Synthesis and Carbonic Anhydrase Inhibitory Activity of Metal Complexes of 4,5-Disubstituted-3-Mercapto-1,2,4-Triazole Derivatives

    Science.gov (United States)

    Scozzafava, Andrea; Cavazza, Christine; Saramet, Ioana; Briganti, Fabrizio; Banciu, Mircea D.

    1998-01-01

    Complexes containing five 4,5-disubstituted-3-mercapto-1,2,4-triazoles and Zn(II), Hg(II) and Cu(I) were synthesized and characterized by standard procedures (elemental analysis; IR, electronic and NMR spectroscopy, conductimetry and TG analysis). Both the thione as well as the thiolate forms of the ligands were evidenced to interact with the metal ions in the prepared complexes. The original mercaptans and their metal complexes behave as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV, but did not lower intraocular pressure in rabbits in animal models of glaucoma. PMID:18475819

  7. Complexes with biologically active ligands. Part 11. Synthesis and carbonic anhydrase inhibitory activity of metal complexes of 4,5-disubstituted-3-mercapto-1,2,4-triazole derivatives.

    Science.gov (United States)

    Scozzafava, A; Cavazza, C; Supuran, C T; Saramet, I; Briganti, F; Banciu, M D

    1998-01-01

    Complexes containing five 4,5-disubstituted-3-mercapto-1,2,4-triazoles and Zn(II), Hg(II) and Cu(I) were synthesized and characterized by standard procedures (elemental analysis; IR, electronic and NMR spectroscopy, conductimetry and TG analysis). Both the thione as well as the thiolate forms of the ligands were evidenced to interact with the metal ions in the prepared complexes. The original mercaptans and their metal complexes behave as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II and IV, but did not lower intraocular pressure in rabbits in animal models of glaucoma.

  8. Formation of local native-like tertiary structures in the slow refolding reaction of human carbonic anhydrase II as monitored by circular dichroism on tryptophan mutants.

    Science.gov (United States)

    Andersson, D; Freskgård, P O; Jonsson, B H; Carlsson, U

    1997-04-15

    In the present study, near-UV CD kinetic measurements on mutants, in which one Trp residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCAII). In addition, the formation of the active site was probed by the binding of a fluorescent sulfonamide inhibitor. The development of the individual Trp CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCAII at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Trp residues 16, 97, and 245 were analyzed. Trp16 probes the N-terminal domain (amino acid residues 1-25), and this part is forming its tertiary structure slower than the major domain (amino acid residues 26-260) of the protein molecule, which contains the active site and a dominating beta-sheet. An essentially native structure of the major domain seems to act as a template for the correct folding of the N terminus. Trp97 is located in a hydrophobic cluster comprising beta-strands 3-5 in the protein core. Previously, we have shown that this region is remarkably stable and compact, and stopped-flow fluorescence data indicate that Trp97 is buried in an apolar compact cluster within a few milliseconds [Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Martensson, L.-G., Gentile, M., Bóren, K., & Carlsson, U. (1995) Biochemistry 34, 8606-8620; Jonasson, P., Aronsson, G., Carlsson, U., & Jonsson, B.-H. (1997) Biochemistry 36 (in press)]. Here it is shown that the development of the native tertiary structure at Trp97 occurs in the minute time domain. Trp245 is located in a long loop between the N-terminal domain and the core structure. Although this Trp has attained native-like fluorescence properties within the dead time of the CD experiment, it assumes a

  9. Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase.

    Science.gov (United States)

    Pinter, Tyler B J; Stillman, Martin J

    2015-11-01

    Mammalian metallothioneins (MTs) bind up to seven Zn(2+) using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7-MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal β-domain [(Zn3Cys9)(3-)] and C-terminal α-domain [(Zn4Cys11)(3-)]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn-βαMT1A (human metallothionein 1A isoform; n=0-7). The apo-CA is shown to compete effectively only with Zn2-3-βMT and Zn4-αMT. Detailed modelling of the ESI mass spectral data have revealed the zinc-binding affinities of each of the zinc-binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities [log(KF)] of the isolated β-domain were: 12.2, 11.7 and 11.4 and the four isolated α-domain affinities were: 13.5, 13.2, 12.7 and 12.6. These data provide guidance in identification of the location of the strongest-bound and weakest-bound zinc in the intact two-domain Zn7βαMT. The β-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn7-βαMT. The α-domain with the highest affinity binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apo-peptide bundle. We propose that stabilization of the intact Zn6-MT and Zn7-MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.

  10. Carbonic anhydrase activators: X-ray crystal structure of the adduct of human isozyme II with L-histidine as a platform for the design of stronger activators.

    Science.gov (United States)

    Temperini, Claudia; Scozzafava, Andrea; Puccetti, Luca; Supuran, Claudiu T

    2005-12-01

    Activation of the carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, and IV with l-histidine and some of its derivatives has been investigated by kinetic and X-ray crystallographic methods. l-His was a potent activator of isozymes I and IV (activation constants in the range of 4-33microM), and a moderate hCA II activator (activation constant of 113microM). Both carboxy- as well as amino-substituted l-His derivatives, such as the methyl ester or the dipeptide carnosine (beta-Ala-His), acted as more efficient activators as compared to l-His. The X-ray crystallographic structure of the hCA II-l-His adduct showed the activator to be anchored at the entrance of the active site cavity, participating in an extended network of hydrogen bonds with the amino acid residues His64, Asn67, and Gln92 and, with three water molecules connecting it to the zinc-bound water. Although the binding site of l-His is similar to that of histamine, the first CA activator for which the X-ray crystal structure has been reported in complex with hCA II (Briganti, F.; Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran, C. T. Biochemistry1997, 36, 10384) there are important differences of binding between the two structurally related activators, since histamine interacts among others with Asn67 and Gln92 (similarly to l-His), but also with Asn62 and not His64, whereas the number of water molecules connecting them to the zinc-bound water is different (two for histamine, three for l-His). Furthermore, the imidazole moieties of the two activators adopt different conformations when bound to the enzyme active site. Since neither the amino- nor carboxy moieties of l-His participate in interactions with amino acid moieties of the active site, they can be derivatized for obtaining more potent activators, with pharmacological applications for the enhancement of synaptic efficacy. This may constitute a novel approach for the treatment of Alzheimer's disease, aging, and other conditions in

  11. A pilot study on potential plasma hypoxia markers in the radiotherapy of non-small cell lung cancer. Osteopontin, carbonic anhydrase IX and vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Ostheimer, C.; Bache, M.; Guettler, A.; Vordermark, D. [Martin-Luther-University Halle-Wittenberg, Department of Radiation Oncology, Halle (Saale) (Germany); Kotzsch, M. [Technical University Dresden, Department of Pathology, Dresden (Germany)

    2014-03-15

    Hypoxic radioresistance plays a critical role in the radiotherapy of cancer and adversely impacts prognosis and treatment response. This prospective study investigated the interrelationship and the prognostic significance of several hypoxia-related proteins in non-small cell lung cancer (NSCLC) patients treated by radiotherapy ± chemotherapy. Pretreatment osteopontin (OPN), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) plasma levels were determined by ELISA in 55 NSCLC (M0) patients receiving 66 Gy curative-intent radiotherapy or chemoradiation. Marker correlation, association with clinicopathological parameters and the prognostic value of a biomarker combination was evaluated. All biomarkers were linearly correlated and linked to different clinical parameters including lung function, weight loss (OPN), gross tumor volume (VEGF) and T stage (CA IX). High OPN (p = 0.03), VEGF (p = 0.02) and CA IX (p = 0.04) values were significantly associated with poor survival. Double marker combination additively increased the risk of death by a factor of 2 and high plasma levels of the triple combination OPN/VEGF/CA IX yielded a 5.9-fold risk of death (p = 0.009). The combined assessment of OPN/VEGF/CA IX correlated independently with prognosis (p = 0.03) in a multivariate Cox regression model including N stage, T stage and GTV. This pilot study suggests that a co-detection augments the prognostic value of single markers and that the integration of OPN, VEGF and CA IX into a hypoxic biomarker profile for the identification of patients with largely hypoxic and radioresistant tumors should be further evaluated. (orig.) [German] Hypoxische Radioresistenz spielt eine kritische Rolle in der Radiotherapie maligner Tumoren und beeinflusst Prognose und Therapieansprechen negativ. Diese prospektive Studie untersuchte den Zusammenhang und die prognostische Bedeutung einiger hypoxieassoziierter Proteine bei Patienten mit nicht-kleinzelligem Bronchialkarzinom

  12. 9,10-Dibromo-N-aryl-9,10-dihydro-9,10-[3,4]epipyrroloanthracene-12,14-diones: Synthesis and Investigation of Their Effects on Carbonic Anhydrase Isozymes I, II, IX, and XII.

    Science.gov (United States)

    Göksu, Haydar; Topal, Meryem; Keskin, Ali; Gültekin, Mehmet S; Çelik, Murat; Gülçin, İlhami; Tanc, Muhammet; Supuran, Claudiu T

    2016-06-01

    N-substituted maleimides were synthesized from maleic anhydride and primary amines. 1,4-Dibromo-dibenzo[e,h]bicyclo-[2,2,2]octane-2,3-dicarboximide derivatives (4a-f) were prepared by the [4+2] cycloaddition reaction of dibromoanthracenes with the N-substituted maleimide derivatives. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory effects of the new derivatives were assayed against the human (h) isozymes hCA I, II, IX, and XII. All tested bicyclo dicarboximide derivatives exhibited excellent inhibitory effects in the nanomolar range, with Ki values in the range of 117.73-232.87 nM against hCA I and of 69.74-111.51 nM against hCA II, whereas they were low micromolar inhibitors against hCA IX and XII. PMID:27174792

  13. Degradation products of the artificial azo dye, Allura red, inhibit esterase activity of carbonic anhydrase II: A basic in vitro study on the food safety of the colorant in terms of enzyme inhibition.

    Science.gov (United States)

    Esmaeili, Sajjad; Ashrafi-Kooshk, Mohammad Reza; Khaledian, Koestan; Adibi, Hadi; Rouhani, Shohre; Khodarahmi, Reza

    2016-12-15

    Allura red is a widely used food colorant, but there is debate on its potential security risk. In the present study, we found that degradation products of the dye were more potent agents with higher carbonic anhydrase inhibitory action than the parent dye. The mechanism by which the compounds inhibit the enzyme activity has been determined as competitive mode. In addition, the enzyme binding properties of the compounds were investigated employing different spectroscopic techniques and molecular docking. The analyses of fluorescence quenching data revealed the existence of the same binding site for the compounds on the enzyme molecule. The thermodynamic parameters of ligand binding were not similar, which indicates that different interactions are responsible in binding of the parent dye and degradation products to the enzyme. It appears that enzyme inhibition should be considered, more seriously, as a new opened dimension in food safety. PMID:27451209

  14. Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells with a Cu(II)-Selective Carbonic Anhydrase-Based Biosensor

    Science.gov (United States)

    McCranor, Bryan J.; Szmacinski, Henryk; Zeng, Hui Hui; Stoddard, A.K.; Hurst, Tamiika; Fierke, Carol A.; Lakowicz, J.R.

    2014-01-01

    Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed. PMID:24671220

  15. Bio-sequestration of CO2 Using Carbonic Anhydrase in situ Encapsulated Inside Electrospun Hollow Fibers%静电纺丝制备中空纤维原位固定化碳酸酐酶用于二氧化碳的吸收

    Institute of Scientific and Technical Information of China (English)

    崔建东; 李莹; 姬晓元; 边红杰; 张羽飞; 苏志国; 马光辉; 张松平

    2014-01-01

    Carbonic anhydrase catalyzed bio-sequestration of CO2 to form HCO-3 , followed by trapping as solid CaCO3 is one of the most promising technologies for CO2 capturing. The effects of reaction condition on the CO2 hydration using free carbonic anhydrase were systematically investigated. In order to improve the stability of the enzyme and facility its recycling, the carbonic anhydrase was in situ encapsulated inside hollow fibers via a novel co-axial electrospinning technology. Compared with the free enzyme, the immobilized carbonic an-hydrase showed much improved thermal stability and suffered much reduced inhibitory effects from cation ions, such as Cu2+ and Fe3+. After 11 reuses, the immobilized enzyme retained about 81. 9% of its original activity by comparing the amount of formed CaCO3 precipitation. In the presence of immobilized carbonic anhydrase, both calcite and vaterite CaCO3 solid were formed;while in the absence of enzyme or with free carbonic anhy-drase, only calcite CaCO3 was observed.%考察了游离碳酸酐酶吸收CO2水合体系反应条件,并通过同轴共纺静电纺丝技术制备出中空结构纤维,实现了碳酸酐酶在中空纤维中的原位包埋,提高了酶的稳定性并便于回收和重复利用.实验结果表明,固定化碳酸酐酶的热稳定性显著增强,受Cu2+和Fe3+等金属离子的抑制作用大幅度降低.连续使用11次后所生成的CaCO3沉淀量仍能达到首次使用的81.9%.固定化酶体系生成的CaCO3沉淀包括方解石型和球文石型2种晶形,而无酶和加入游离碳酸酐酶的反应体系则主要生成方解石型CaCO3沉淀.

  16. Nitrogen control of chloroplast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, G.W.

    1992-07-01

    This project is directed toward understanding how the availability of nitrogen affects the accumulation of chloroplast pigments and proteins functioning in energy transduction and carbon metabolism. Molecular analyses performed with Chlamydomonas reinhardtii grown in a continuous culture system such that ammonium concentration is maintained at a low steady-state concentration so as to limit cell division. As compared to chloroplasts from cells of non-limiting nitrogen provisions, chloroplasts of N-limited cells are profoundly chlorophyll-deficient but still assimilate carbon for deposition of as starch and as storage lipids. Chlorophyll deficiency arises by limiting accumulation of appropriate nuclear-encoded mRNAs of and by depressed rates of translation of chloroplast mRNAs for apoproteins of reaction centers. Chloroplast translational effects can be partially ascribed to diminished rates of chlorophyll biosynthesis in N-limited cells, but pigment levels are not determinants for expression of the nuclear light-harvesting protein genes. Consequently, other signals that are responsive to nitrogen availability mediate transcriptional or post-transcriptional processes for accumulation of the mRNAs for LHC apoproteins and other mRNAs whose abundance is dependent upon high nitrogen levels. Conversely, limited nitrogen availability promotes accumulation of other proteins involved in carbon metabolism and oxidative electron transport in chloroplasts. Hence, thylakoids of N-limited cells exhibit enhanced chlororespiratory activities wherein oxygen serves as the electron acceptor in a pathway that involves plastoquinone and other electron carrier proteins that remain to be thoroughly characterized. Ongoing and future studies are also outlined.

  17. The role of extracellular carbonic anhydrase activity in inorganic carbon utilization of Phaeocystis globosa (Prymnesiophyceae) : A comparison with other marine algae using the isotopic disequilibrium technique

    NARCIS (Netherlands)

    Elzenga, JTM; Prins, HBA; Stefels, J

    2000-01-01

    The utilization of inorganic carbon species by the marine microalga Phaeocystis globasa (Prymnesiophyceae) and several other algal species from different taxa, was investigated by determining the time course of C-14 incorporation in isotopic disequilibrium experiments. From these kinetic data, concl

  18. The plant carbonic anhydrase at karst area and its ecological effects%植物碳酸酐酶对岩溶作用的影响及其生态效应

    Institute of Scientific and Technical Information of China (English)

    李强; 何媛媛; 曹建华; 梁建宏; 朱敏洁

    2011-01-01

    广泛存在各种类型生物细胞中的碳酸酐酶(CA),通过催化CO2和HCO3之间的相互转化,驱动岩溶过程.文章通过分析我国西南典型岩溶区植物叶片和根系CA活性,探讨其与岩溶作用的相互关系,结果表明:岩溶生态系统中植物的碳酸酐酶活性差异较大,并在植物的生长期发生变动,植株根系CA活性>成熟叶片CA活性,因而在土壤水分充足的条件下,根系分泌的CA催化CO2+H2O(←→)HCO3-+H+过程,促进石灰岩溶解,加快成土速率,并通过固定根呼吸和土壤微生物分解所释放的CO2产生岩溶碳汇效应;非岩溶生态系统由于土壤碳酸钙含量低,造成植物根系CA表达活性较低.%C02 as the important driving force in karstification process, Carbonic anhydrase (CA) can quickly catalyses the reversible interconversion of carbon dioxide and bicarbonate. Typical karst region in Southwest China was selected to investigate the relationship between Carbonic anhydrase of plant and karstification. The results shows that plant CA activity changes with living period and seasons and have the sequence just as root >mature leaves. The high CA activity in root at karst ecosystem indicates that CA in plant can quickly catalyses the reversible interconversion of carbon dioxide and bicarbonate when the soil has sufficient water. It will accelerate the dissolution of limestone and improve the rate of soil rock weathering. In turn, the nutrients released from limestone will improve plant growth and metabolism. It also can produce the CO2 sink from soil. Moreover, at non-karst area, the plants don't need to express high CA activity due to this kind of soil lake of limestone.

  19. Over-expression of the β-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content.

    Science.gov (United States)

    Long, Benedict M; Rae, Benjamin D; Badger, Murray R; Price, G Dean

    2011-09-01

    Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.

  20. Close association of carbonic anhydrase (CA2a & CA15a, Na+/H+ exchanger (Nhe3b, and ammonia transporter Rhcg1 in zebrafish ionocytes responsible for Na+ uptake

    Directory of Open Access Journals (Sweden)

    Yusuke eIto

    2013-04-01

    Full Text Available Freshwater fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs on the skin epithelium of zebrafish larvae (Danio rerio are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were up-regulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell-surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morphorino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b, and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+ uptake under freshwater conditions by forming a transport metabolon with Nhe3b.

  1. Chloroplast Redox Poise

    DEFF Research Database (Denmark)

    Steccanella, Verdiana

    The redox state of the chloroplast is maintained by a delicate balance between energy production and consumption and is affected by the need to avoid increased production of reactive oxygen species (ROS). Redox power and ROS generated in the chloroplast are essential for maintaining physiological...... metabolic pathways and for optimizing chloroplast functions. The redox poise of photosynthetic electron transport components like plastoquinone is crucial to initiate signaling cascades and might also be involved in key biosynthetic pathways such as chlorophyll biosynthesis. We, therefore, explored...

  2. Nitrogen control of chloroplast development: Progress report

    International Nuclear Information System (INIS)

    A manifestation of nitrogen deficiency in vascular plants and algae is chlorosis, indicating that chloroplast biogenesis can be strongly restricted by direct or indirect effects of nitrogen assimilation products. To define the molecular basis of nitrogen responses we are using Chlamydomonas reinhardtii. Depending on the levels of ammonium, steady-state deficiency conditions are established such that the cellular levels of chlorophylls and xanthophylls are depressed. Chloroplasts in nitrogen-deficient cells contain appreciable levels of carbon assimilation enzyme and thylakoids with high electron transport activities. However, the light harvesting complexes are nearly absent and Photosystem I exhibits unusual characteristics. Studies of rates of protein synthesis by in vivo pulse-chase labeling and levels of RNAs encoded by the chloroplast and nuclear genomes have been initiated: the accumulation of transcripts for the nuclear light-harvesting apoproteins is dramatically altered qualitatively and quantitatively; there is no major effect on chloroplast RNAs but, in general, these are inefficiently utilized for protein synthesis until nitrogen is provided to the cultures. Supplying nitrogen results in an almost immediate release of chloroplast mRNAs from a translational arrest but the stimulation of the accumulation of nuclear transcripts for light-harvesting apoproteins does not occur until after a 1-2 hour lag

  3. Gene cloning, expression and enzyme activity analysis of the carbonic anhydrase from Porphyra haitanensis (Rhodophyta)%坛紫菜碳酸酐酶基因的克隆、表达及酶活性分析

    Institute of Scientific and Technical Information of China (English)

    郇丽; 贾兆君; 张宝玉; 牛建峰; 林阿朋; 何林文; 王广策

    2014-01-01

    以坛紫菜丝状体为材料,采用RACE方法获得坛紫菜碳酸酐酶(CA)基因的全长cDNA。该cDNA全长1081 bp,具有一个825 bp的开放阅读框,可编码274个氨基酸。序列同源性分析显示该cDNA序列推导的氨基酸序列与其他物种的碳酸酐酶具有较高的一致性,其中与条斑紫菜的一致性达到96%。氨基酸序列分析表明该蛋白为β-CA,含有两个CA活性位点,无跨膜结构,可能存在一个信号肽将其定位到叶绿体中,与藻类和细菌聚类。原核诱导表达得到一个72 kDa左右的融合蛋白,酶活测定结果显示此蛋白具有碳酸酐酶活性。该实验对进一步深入研究坛紫菜 CA 的功能及坛紫菜碳代谢、光合作用等生理过程具有重要的参考价值。%Carbonic anhydrase (CA), a zinc-containing enzyme is widespread in living organisms, catalyses the reversible hydration of CO2 and 3HCO-. In this study, a full-length cDNA of CA was isolated from Porphyra haitanensis with rapid amplification of cDNA ends (RACE). This sequence was 1 081 bp in length and encodes a polypeptide of 274 amino acid residues. The deduced polypeptide showed high identities with the CA genes ranging from unicellular algae and bacteria to green plant. Phylogenetic tree analysis showed that the CA gene from P. haitanensis was more closely assembled with algae and bacteria. A ~72 kDa fused protein was obtained by the recombinant prokaryotic expression and the enzyme activity analysis showed that it had the activity of CA.

  4. 苯磺酰胺从碳酸酐酶II中脱离过程的分子动力学模拟%Molecular Dynamics Simulations of the Unbinding of Phenylsulfonamide from Carbonic Anhydrase II

    Institute of Scientific and Technical Information of China (English)

    孙维琦; 张继龙; 郑清川; 孙志伟; 张红星

    2013-01-01

      综合运用分子动力学模拟和自由能计算方法研究了苯磺酰胺分子从碳酸酐酶II (CA II)的活性位点脱离过程中底物与酶之间的动态相互作用。脱离过程的平均力势(PMF)显示,底物脱离时存在一个特殊的结合状态。其中,静电相互作用占据了主导地位。轨迹分析显示,除了金属离子的配位作用之外,底物脱离路径上的关键残基Leu198、Thr199和Thr200通过与底物磺胺基的氢键作用阻碍了底物从酶中的脱离。当前的研究对于深入认识磺胺类药物与CA II的详细结合过程和相关的药物改良与设计具有重要的指导意义。%Molecular dynamics (MD) simulations and free energy calculations were integrated to investigate substrate-enzyme dynamic interactions during the unbinding of phenylsulfonamide from carbonic anhydrase II (CA II). The potential of mean force (PMF) along the unbinding pathway shows that a special ligand-binding state exists, and the electrostatic interaction dominates the ligandʹs binding with CA II. The analysis of trajectories reveals that, apart from the zinc ion, the key residues in the unbinding pathway, Leu198, Thr199, and Thr200, prevent the substrateʹs unbinding from the enzyme by hydrogen bonding with the sulfanilamide group of the substrate. The present results are of direct significance for the in-depth understanding of the sulfonamide-CA II binding process and related drug design.

  5. Extra and intracelular activities of carbonic anhydrase of the marine microalga Tetraselmis gracilis (Chlorophyta Atividade extra e intracelular da Anidrase Carbônica na microalga marinha Tetraselmis gracilis (Chlorophyta

    Directory of Open Access Journals (Sweden)

    Marilda Rigobello-Masini

    2003-07-01

    Full Text Available The activities of extra and intracellular carbonic anhydrases (CA were studied in the microalgae Tetraselmis gracilis (Kylin Butcher (Chlorophyta, Prasinophyceae growing in laboratory cultivation. During ten days of batch cultivation, daily determinations of pH, cell number, enzymatic activity, and total dissolved inorganic carbon (DIC, as well as its main species, CO2 and HCO3-, were performed. Enzymatic activity increased as the growing cell population depleted inorganic carbon from the medium. Carbon dioxide concentration decreased quickly, especially in the third day of cultivation, when a significant increase of the intracellular enzymatic activity was observed. Bicarbonate concentration had its largest decrease in the cultivation medium in the fourth day, when the activity of the extracellular enzyme had its largest increase, suggesting its use by the alga through CA activity. After the fourth cultivation day, half of the cultures were aerated with CO2-free atmospheric air, which caused an increase in the total and external activity of the enzyme, although, in this condition, the stationary growth phase began earlier than in cultures aerated with atmospheric air. The pH of the media was measured daily, increasing from the first to the fourth day, and remaining almost constant until the end of the cultivation. Algal material transferred to the dark lost all enzymatic activity.As atividades da Anidrase Carbônica (AC extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin Butcher (Chlorophyta, Prasinophyceae crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinações diárias do pH, número de células, atividades enzimáticas, carbono inorgânico total dissolvido (CID e suas principais espécies CO2 e HCO3- foram feitas. A atividade enzimática aumentou na medida em que a população celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentração de dióxido de

  6. Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.

    Science.gov (United States)

    Atkinson, Nicky; Feike, Doreen; Mackinder, Luke C M; Meyer, Moritz T; Griffiths, Howard; Jonikas, Martin C; Smith, Alison M; McCormick, Alistair J

    2016-05-01

    Many eukaryotic green algae possess biophysical carbon-concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H(14) CO3 (-) uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild-type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species. PMID:26538195

  7. Interactive Effect of GA3, N and P Ameliorate Growth, Seed and Fibre Yield by Enhancing Photosynthetic Capacity and Carbonic Anhydrase Activity of Linseed:A Dual Purpose Crop

    Institute of Scientific and Technical Information of China (English)

    Mohammad N Khan; Firoz Mohammad

    2013-01-01

    Linseed (Linum usitatissimum L.) is an important dual-purpose, industrial crop. Its seeds are used for the extraction of oil and stem for fibres. However, the production of linseed is not going parallel with the increasing demand of its products. The present work was carried out with an aim to find out whether exogenous application of gibberellic acid (GA3) with or without graded levels of nitrogen (N) and phosphorus (P) could improve the performance of three linseed genotypes Parvati, Shekhar and Shubhra together with minimizing the costly fertilizer input and losses. Four combinations of N and P, viz., 0 mg N+0 mg P kg-1 soil (N0P0), N13.4P4.46, N26.8P8.94 and N40.2P13.4 were constituted. Half dose of each combination was applied basally at the time of sowing and remaining half dose was given at 40 d after sowing (DAS) as foliar spray along with 10-6 mol L-1 GA3. Prior to sowing, the seeds of each linseed genotype were grouped in to two, one group of seeds was soaked in 0 mol L-1 GA3 (control) and the other group was soaked in 10-6 M GA3 solution, each for 8 hours. Treatments were comprised of (i) 0 mol L-1 GA3+N0P0 (T0, control), (ii) 10-6 mol L-1 GA3+N13.4P4.46 (T1), (iii) 10-6 mol L-1 GA3+N26.8P8.94 (T2) and (iv) 10-6 mol L-1 GA3+N40.2P13.4 (T3). The crop performance was assessed in terms of growth, physiological and biochemical parameters at 60 and 75 DAS and yield attributes at harvest (175 DAS). The results showed a parallel increase in most of the parameters with increasing levels of N and P. However, application of 10-6 mol L-1 GA3 in association with N26.8P8.94 proved best, it enhanced seed yield, oil yield and fibre yield by 83.3, 97.3 and 78.7%, respectively accompanied with increase in net photosynthetic rate, carbonic anhydrase activity and dry matter accumulation. Among the genotypes tested, Shubhra performed best, while Parvati the least for most of the parameters studied. Thus, combined application of 10-6 mol L-1 GA3 plus N26.8P8.94 proved best

  8. Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation

    OpenAIRE

    Stettler, Michaela; Eicke, Simona; Mettler, Tabea; Messerli, Gaëlle; Hörtensteiner, Stefan; Zeeman, Samuel C.

    2009-01-01

    In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels...

  9. 血清抗碳酸酐酶Ⅲ抗体ELISA检测方法的建立与初步应用%Establishment and preliminary application of the ELISA method for anti-carbonic anhydrase III antibody detection

    Institute of Scientific and Technical Information of China (English)

    刘辰庚; 王培昌

    2011-01-01

    目的 建立人血清抗碳酸酐酶(CA)Ⅲ抗体的ELISA检测方法,并对系统性红斑狼疮、皮肌炎、糖尿病肾病、高血压肾病患者和健康人群的血清抗CAⅢ抗体水平进行初步调查.方法 使用抗CAⅢ抗体标准品、CAⅢ及相应酶标抗体建立血清抗CAⅢ抗体ELISA检测方法,验证试剂稳定性、标本保存稳定性,并进行精密度、灵敏度、回收率、抗干扰性等方法学评价;各项技术指标均合格后对系统性红斑狼疮、皮肌炎、糖尿病肾病和高血压肾病患者的血清进行抗CAⅢ抗体水平检测.结果 成功建立ELISA检测人血清抗CAⅢ抗体的方法,其批内精密度为6.2%,批间精密度为8.2%,灵敏度为0.025,回收率为106%,且具有较好的抗干扰性、试剂稳定性和标本保存稳定性.系统性红斑狼疮和糖尿病肾病患者的血清抗CAⅢ抗体水平高于健康对照相(P<0.05),阳性率分别为43%和18%.皮肌炎和高血压肾病患者的血清抗CAⅢ抗体水平与健康对照组比较无统计学差异(P>0.05),且未出现阳性结果.结论 使用现有市售试剂进行人血清抗CAⅢ抗体的ELISA检测是可行的,抗CAⅢ抗体可能参与了系统性红斑狼疮和糖尿病肾病的发生发展.%Objective To establish an ELISA method for anti-carbonic anhydrase III (CA III ) antibody detection, and to evaluate the serum level of anti-CA III antibody in normal control group and patients with systemic lupus erythemato-sus, dermatomyositis, 2-type diabetic nephropathy and hypertensive nephropathy respectively. Methods To establish the ELISA method using CA III, anti-CA III antibody and enzyme labeled secondary antibody. To evaluate the stability of the regent and sample, and the sensitivity, stability and anti-interference performance of the ELISA method. To investigate the anti-CA III antibody level in serum of normal control group and patients with systemic lupus erythematosus, dermatomyositis, 2-type

  10. SwissProt search result: AK060777 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060777 001-033-C01 (P17067) Carbonic anhydrase, chloroplast precursor (EC 4.2.1.1) (Carbon...ate dehydratase) [Contains: Carbonic anhydrase, 27 kDa isoform; Carbonic anhydrase, 25 kDa isoform] CAHC_PEA 2e-44 ...

  11. Genetic Analysis of Chloroplast Translation

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, Alice

    2005-08-15

    The assembly of the photosynthetic apparatus requires the concerted action of hundreds of genes distributed between the two physically separate genomes in the nucleus and chloroplast. Nuclear genes coordinate this process by controlling the expression of chloroplast genes in response to developmental and environmental cues. However, few regulatory factors have been identified. We used mutant phenotypes to identify nuclear genes in maize that modulate chloroplast translation, a key control point in chloroplast gene expression. This project focused on the nuclear gene crp1, required for the translation of two chloroplast mRNAs. CRP1 is related to fungal proteins involved in the translation of mitochondrial mRNAs, and is the founding member of a large gene family in plants, with {approx}450 members. Members of the CRP1 family are defined by a repeated 35 amino acid motif called a ''PPR'' motif. The PPR motif is closely related to the TPR motif, which mediates protein-protein interactions. We and others have speculated that PPR tracts adopt a structure similar to that of TPR tracts, but with a substrate binding surface adapted to bind RNA instead of protein. To understand how CRP1 influences the translation of specific chloroplast mRNAs, we sought proteins that interact with CRP1, and identified the RNAs associated with CRP1 in vivo. We showed that CRP1 is associated in vivo with the mRNAs whose translation it activates. To explore the functions of PPR proteins more generally, we sought mutations in other PPR-encoding genes: mutations in the maize PPR2 and PPR4 were shown to disrupt chloroplast ribosome biogenesis and chloroplast trans-splicing, respectively. These and other results suggest that the nuclear-encoded PPR family plays a major role in modulating the expression of the chloroplast genome in higher plants.

  12. Nitrogen control of chloroplast differentiation. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, G.W.

    1992-07-01

    This project is directed toward understanding how the availability of nitrogen affects the accumulation of chloroplast pigments and proteins functioning in energy transduction and carbon metabolism. Molecular analyses performed with Chlamydomonas reinhardtii grown in a continuous culture system such that ammonium concentration is maintained at a low steady-state concentration so as to limit cell division. As compared to chloroplasts from cells of non-limiting nitrogen provisions, chloroplasts of N-limited cells are profoundly chlorophyll-deficient but still assimilate carbon for deposition of as starch and as storage lipids. Chlorophyll deficiency arises by limiting accumulation of appropriate nuclear-encoded mRNAs of and by depressed rates of translation of chloroplast mRNAs for apoproteins of reaction centers. Chloroplast translational effects can be partially ascribed to diminished rates of chlorophyll biosynthesis in N-limited cells, but pigment levels are not determinants for expression of the nuclear light-harvesting protein genes. Consequently, other signals that are responsive to nitrogen availability mediate transcriptional or post-transcriptional processes for accumulation of the mRNAs for LHC apoproteins and other mRNAs whose abundance is dependent upon high nitrogen levels. Conversely, limited nitrogen availability promotes accumulation of other proteins involved in carbon metabolism and oxidative electron transport in chloroplasts. Hence, thylakoids of N-limited cells exhibit enhanced chlororespiratory activities wherein oxygen serves as the electron acceptor in a pathway that involves plastoquinone and other electron carrier proteins that remain to be thoroughly characterized. Ongoing and future studies are also outlined.

  13. Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize.

    Directory of Open Access Journals (Sweden)

    Prakitchai Chotewutmontri

    2016-07-01

    Full Text Available Chloroplast genomes in land plants contain approximately 100 genes, the majority of which reside in polycistronic transcription units derived from cyanobacterial operons. The expression of chloroplast genes is integrated into developmental programs underlying the differentiation of photosynthetic cells from non-photosynthetic progenitors. In C4 plants, the partitioning of photosynthesis between two cell types, bundle sheath and mesophyll, adds an additional layer of complexity. We used ribosome profiling and RNA-seq to generate a comprehensive description of chloroplast gene expression at four stages of chloroplast differentiation, as displayed along the maize seedling leaf blade. The rate of protein output of most genes increases early in development and declines once the photosynthetic apparatus is mature. The developmental dynamics of protein output fall into several patterns. Programmed changes in mRNA abundance make a strong contribution to the developmental shifts in protein output, but output is further adjusted by changes in translational efficiency. RNAs with prioritized translation early in development are largely involved in chloroplast gene expression, whereas those with prioritized translation in photosynthetic tissues are generally involved in photosynthesis. Differential gene expression in bundle sheath and mesophyll chloroplasts results primarily from differences in mRNA abundance, but differences in translational efficiency amplify mRNA-level effects in some instances. In most cases, rates of protein output approximate steady-state protein stoichiometries, implying a limited role for proteolysis in eliminating unassembled or damaged proteins under non-stress conditions. Tuned protein output results from gene-specific trade-offs between translational efficiency and mRNA abundance, both of which span a large dynamic range. Analysis of ribosome footprints at sites of RNA editing showed that the chloroplast translation machinery

  14. Chloroplast ribosomes and protein synthesis.

    OpenAIRE

    Harris, E. H.; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles i...

  15. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

    Science.gov (United States)

    Nielsen, Agnieszka Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos; Wlodarczyk, Artur Jacek; Gnanasekaran, Thiyagarajan; Perestrello Ramos H de Jesus, Maria; King, Brian Christopher; Bakowski, Kamil; Jensen, Poul Erik

    2016-07-01

    Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. PMID:27005523

  16. Chloroplast in Plant-Virus Interaction

    Science.gov (United States)

    Zhao, Jinping; Zhang, Xian; Hong, Yiguo; Liu, Yule

    2016-01-01

    In plants, the chloroplast is the organelle that conducts photosynthesis. It has been known that chloroplast is involved in virus infection of plants for approximate 70 years. Recently, the subject of chloroplast-virus interplay is getting more and more attention. In this article we discuss the different aspects of chloroplast-virus interaction into three sections: the effect of virus infection on the structure and function of chloroplast, the role of chloroplast in virus infection cycle, and the function of chloroplast in host defense against viruses. In particular, we focus on the characterization of chloroplast protein-viral protein interactions that underlie the interplay between chloroplast and virus. It can be summarized that chloroplast is a common target of plant viruses for viral pathogenesis or propagation; and conversely, chloroplast and its components also can play active roles in plant defense against viruses. Chloroplast photosynthesis-related genes/proteins (CPRGs/CPRPs) are suggested to play a central role during the complex chloroplast-virus interaction. PMID:27757106

  17. Biosynthesis of starch in chloroplasts.

    Science.gov (United States)

    Nomura, T; Nakayama, N; Murata, T; Akazawa, T

    1967-03-01

    The enzymic synthesis of ADP-glucose and UDP-glucose by chloroplastic pyrophosphorylase of bean and rice leaves has been demonstrated by paper chromatographic techniques. In both tissues, the activity of UDP-glucose-pyrophosphorylase was much higher than ADP-glucose-pyrophosphorylase. Glycerate-3-phosphate, phosphoenolpyruvate and fructose-1,6-diphosphate did not stimulate ADP-glucose formation by a pyrophosphorylation reaction. The major metabolic pathway for UDP-glucose utilization appears to be the synthesis of either sucrose or sucrose-P. On the other hand, a specific precursor role of ADP-glucose for synthesizing chloroplast starch by the ADP-glucose-starch transglucosylase reaction is supported by the coupled enzyme system of ADP-glucose-pyrophosphorylase and transglucosylase, isolated from chloroplasts. None of the glycolytic intermediates stimulated the glucose transfer in the enzyme sequence of reaction system employed. PMID:4292567

  18. Mesophyll Chloroplast Investment in C3, C4 and C2 Species of the Genus Flaveria.

    Science.gov (United States)

    Stata, Matt; Sage, Tammy L; Hoffmann, Natalie; Covshoff, Sarah; Ka-Shu Wong, Gane; Sage, Rowan F

    2016-05-01

    The mesophyll (M) cells of C4 plants contain fewer chloroplasts than observed in related C3 plants; however, it is uncertain where along the evolutionary transition from C3 to C4 that the reduction in M chloroplast number occurs. Using 18 species in the genus Flaveria, which contains C3, C4 and a range of C3-C4 intermediate species, we examined changes in chloroplast number and size per M cell, and positioning of chloroplasts relative to the M cell periphery. Chloroplast number and coverage of the M cell periphery declined in proportion to increasing strength of C4 metabolism in Flaveria, while chloroplast size increased with increasing C4 cycle strength. These changes increase cytosolic exposure to the cell periphery which could enhance diffusion of inorganic carbon to phosphenolpyruvate carboxylase (PEPC), a cytosolic enzyme. Analysis of the transcriptome from juvenile leaves of nine Flaveria species showed that the transcript abundance of four genes involved in plastid biogenesis-FtsZ1, FtsZ2, DRP5B and PARC6-was negatively correlated with variation in C4 cycle strength and positively correlated with M chloroplast number per planar cell area. Chloroplast size was negatively correlated with abundance of FtsZ1, FtsZ2 and PARC6 transcripts. These results indicate that natural selection targeted the proteins of the contractile ring assembly to effect the reduction in chloroplast numbers in the M cells of C4 Flaveria species. If so, efforts to engineer the C4 pathway into C3 plants might evaluate whether inducing transcriptome changes similar to those observed in Flaveria could reduce M chloroplast numbers, and thus introduce a trait that appears essential for efficient C4 function.

  19. Evolution of chloroplast vesicle transport.

    Science.gov (United States)

    Westphal, Sabine; Soll, Jürgen; Vothknecht, Ute C

    2003-02-01

    Vesicle traffic plays a central role in eukaryotic transport. The presence of a vesicle transport system inside chloroplasts of spermatophytes raises the question of its phylogenetic origin. To elucidate the evolution of this transport system we analyzed organisms belonging to different lineages that arose from the first photosynthetic eukaryote, i.e. glaucocystophytes, chlorophytes, rhodophytes, and charophytes/embryophytes. Intriguingly, vesicle transport is not apparent in any group other than embryophytes. The transfer of this eukaryotic-type vesicle transport system from the cytosol into the chloroplast thus seems a late evolutionary development that was acquired by land plants in order to adapt to new environmental challenges.

  20. Influence of topical carbonic anhydrase inhibitor on the expression of aquaporin-1 in rat cornea with neovascularization%碳酸酐酶抑制剂的局部应用对大鼠角膜新生血管形成过程中水通道蛋白1表达的影响

    Institute of Scientific and Technical Information of China (English)

    张洁; 李立

    2011-01-01

    (t=2.48,P=0.02),2个组AQP1灰度值分别为88.01±11.03和58.10±12.14,差异有统计学意义(t=9.99,P=0.00).结论 布林佐胺滴眼液能抑制大鼠角膜碱烧伤后CNV形成过程中AQP1的高表达,从而间接影响VEGF的表达,抑制或延缓CNV的形成.%Background Researches showed that aquaporin-1 (AQP1) is closely associated with corneal neovescularization(CNV).Carbonic anhydrase inhibitor has the inhibitory effect on the AQP1 and further suppresses the CNV.However,the systemic adverse effect of Carbonic anhydrase inhibitor limit its clinical application.Therefore,the influence of topical carbonic anhydrase inhibitor on CNV is concerned.Objective Present study was to investigate the effects of topical carbonic anhydrase inhibitors on the expression of AQP1 in rat cornea after alkali burn and explore its role in corneal neovascularization (CNV).Methods The alkali-burn animal models were established in 60 eyes of 30 clean Sprague Dawley rats by putting the filter paper soaked 1 mol/L NaOH solution at the central cornea for 40 seconds.1% Brinzolamide was topically administered in the 30 eyes of 15 models (Brinzolamide group),and the normal saline solution was used at the same way in other 30 eyes of 15 rats (model group).The 10 eyes of 5 normal Sprague Dawley received the eye drops of normal saline solution as the normal control group.The corneal burning degree was graded on the Mahoney ' s criteria in the third day,and Ee ' s method was used to score the opacification of cornea and the CNV area was analyzed in 3,5,7,10 days under the slit lamp microscope.The cornea tissue was obtained in the tenth day after burning for the observation of the pathology under the light microscope and the ultrastructure under the transmission electron microscope.The expressions of AQP1 and vascular endothelial growth factor(VEGF) in cornea tissue were detected using immunohistochemistry.The use of animals complied with the Statement of ARVO.Results No significant

  1. Free radical generation and antioxidant content in chloroplasts from soybean leaves expsoed to ultraviolet-B

    Energy Technology Data Exchange (ETDEWEB)

    Galatro, A.; Simontacchi, M.; Puntarulo, S. [Univ. of Buenos Aires, School of Pharmacy and Biochemistry, Physical Chemistry, Buenos Aires (Argentina)

    2001-07-01

    The aim of this work was to study the effect of ultraviolet-B (UV-B) exposure on oxidative status in chloroplasts isolated from soybean (Glycine max cv. Hood). Chloroplasts were isolated from soybean leaves excised from either control seedlings or those exposed to 30 and 60 kJ m{sup -2} day{sup -1} of UV-B radiation for 4 days. Chloroplastic oxidative conditions were assessed as carbon-centered radical, carbonyl groups and ascorbyl radical content. Treatment with UV-B increased the carbon-centered radical-dependent EPR signal significantly by 55 and 100% in chloroplasts from leaves exposed to 30 and 60 kJ m{sup -2} day{sup -1} UV-B, respectively, compared to radical content in chloroplasts from control leaves. The content of carbonyl groups increased by 37 and 62% in chloroplasts isolated from soybean leaves irradiated for 4 days with 30 and 60 kJ m{sup -2} day{sup -1} UV-B, respectively. The content of soluble metabolites in isolated chloroplasts should not be taken as absolute in vivo values; however, these data are valuable for comparative studies. UV-B exposure did not significantly affect ascorbyl radical content compared to controls. The content of ascorbic acid and thiols in chloroplasts isolated from leaves exposed to 60 kJ m{sup -2} day{sup -1} UV-B was increased by 117 and 20.8%, respectively, compared to controls. Neither the content of total carotene nor that of {beta}-carotene or {alpha}-tocopherol was affected by the irradiation. The results: presented here suggest that the increased content of lipid radicals and oxidized proteins in the chloroplasts isolated from leaves exposed to UV-B could be ascribed to both the lack of antioxidant response in the lipid soluble fraction and the modest increase in the soluble antioxidant content. (au)

  2. Photosynthesis of root chloroplasts developed in Arabidopsis lines overexpressing GOLDEN2-LIKE transcription factors.

    Science.gov (United States)

    Kobayashi, Koichi; Sasaki, Daichi; Noguchi, Ko; Fujinuma, Daiki; Komatsu, Hirohisa; Kobayashi, Masami; Sato, Mayuko; Toyooka, Kiminori; Sugimoto, Keiko; Niyogi, Krishna K; Wada, Hajime; Masuda, Tatsuru

    2013-08-01

    In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO₂ fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants.

  3. 甘油果糖联合碳酸酐酶抑制剂对高眼压大鼠眼睫状体水通道蛋白1表达的影响%The influence of glyc-fructose combined with carbonic anhydrase inhibitor on the expression of AQP1 in rat eyes

    Institute of Scientific and Technical Information of China (English)

    盛毅; 金丽; 王进; 孙哲

    2015-01-01

    Objective To observe the effect of glyc-fructose combined with carbonic anhydrase inhibitor (CAI) on the expression of AQP 1 in rat eyes.Methods The model of intraocular hypertension in rats were established,and intervention on intraocular hypertension model rats were performed using glycerol fructose and carbonic anhydrase inhibitors.The expression of AQP 1 in the chamber angle tissue was detected in the mRNA and protein level.Results The expression of AQP 1 in the intraocular hypertension group (1,6,24,48 and 72 h) was significantly higher than those in the control group (1.55 ± 0.02,2.22±0.03,2.46 ±0.02,1.88 ±0.04,1.44±0.03; 1.21 ±0.02,3.58 ±0.03,3.81 ± 0.02,4.28 ± 0.04,4.44 ± 0.03,all P < 0.05).Carbonic anhydrase inhibitors could inhibit the expression of AQP 1 in the chamber angle tissue of the intraocular hypertension model rats (intraocular hypertension group vs.CAI group:1.41 ±0.02 vs.1.24 ±0.04; 4.41 ±0.02 vs.2.31 ± 0.04,all P < 0.05).The combined use of glyc-fructose with CAI could inhibit the expression more obviously(intraocular hypertension group vs.Glyc-fructose combined with CAI group:1.41 ± 0.02 vs.1.08±0.03; 4.41 ±0.02 vs.1.47 ±0.03,all P <0.05).Conclusion The expression of AQP1 was elevated in the intraocular hypertension group,and co-administrated with glycerol fructose and brinzolamide could inhibit the expression.%目的 观察联合应用甘油果糖和碳酸酐酶抑制剂对急性高眼压大鼠眼组织水通道蛋白1(AQP1)表达的影响.方法 建立高眼压大鼠模型,并使用甘油果糖和碳酸酐酶抑制剂对高眼压大鼠模型鼠进行干预,检测房角组织AQP1的基因及蛋白表达水平.结果 高眼压大鼠房角组织AQP1的基因和蛋白表达水平(造模后1、6、24、48、72 h:1.55±0.02、2.22±0.03、2.46±0.02、1.88±0.04、1.44±0.03;1.21±0.02、3.58±0.03、3.81±0.02、4.28±0.04、4.44±0.03)均显著高于对照组(1.00±0.00、1.00±0.00,P均<0.05).碳酸酐酶抑制剂

  4. Mutational dynamics of aroid chloroplast genomes.

    Science.gov (United States)

    Ahmed, Ibrar; Biggs, Patrick J; Matthews, Peter J; Collins, Lesley J; Hendy, Michael D; Lockhart, Peter J

    2012-01-01

    A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations. Although similar observations have also been made for chloroplast DNA, genome-wide associations have not been reported. We determined the chloroplast genome sequences for two morphotypes of taro (Colocasia esculenta; family Araceae) and compared these with four publicly available aroid chloroplast genomes. Here, we report the extent of genome-wide association between direct and inverted repeats, indels, and substitutions in these aroid chloroplast genomes. We suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution. PMID:23204304

  5. Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants.

    Science.gov (United States)

    Stata, Matt; Sage, Tammy L; Rennie, Troy D; Khoshravesh, Roxana; Sultmanis, Stefanie; Khaikin, Yannay; Ludwig, Martha; Sage, Rowan F

    2014-11-01

    The evolution of C(4) photosynthesis from C(3) ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C(3), C(3) -C(4) intermediate and C(4) species from 12 lineages of C(4) evolution. All C(3) species had more chloroplasts per M cell area than their C(4) relatives in high-light growth conditions. C(3) species also had higher chloroplast coverage of the M cell periphery than C(4) species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C(4) lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C(4) species than their C(3) relatives. C(3) -C(4) intermediate species generally exhibited similar patterns as their C(3) relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C(4) function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C(4) M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C(4) photosynthesis.

  6. Temperature stability of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] in the form of a solution or in the lyophilized form during storage at -80 °C, 4 °C, 25 °C and 37 °C or pasteurization at 70 °C.

    Science.gov (United States)

    Bian, Y Z; Guo, C; Chang, T M S

    2016-01-01

    Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-min 30 mm Hg Mean Arterial Pressure (MAP) sustained hemorrhagic shock rat model shows that it is more effective than blood in the lowering of elevated intracellular pCO2, recovery of ST-elevation and histology of the heart and intestine. This paper is to analyze the storage and temperature stability. Allowable storage time for RBC is about 1 d at room temperature and 42 d at 4 °C. Also, RBC cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for 1 year even at room temperature. However, Poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. Results of storage stability show that lyophilization extends the storage time to 1 year at 4 °C and 40 d at room temperature (compared to respectively, 42 d and 1 d for RBC). After the freeze-dry process, the enzyme activities of Poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD and 93 ± 3.5% for CAT. After heat pasteurization at 70 °C for 2 h, lyophilized Poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA 97 ± 4%, SOD 100 ± 2.5% and CAT 63.8 ± 4%. More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of Poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving in suitable solutions that continues to have good storage stability though less than that for the lyophilized form. According to the P50 value, Poly-[SFHb-SOD-CAT-CA] retains its

  7. Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX).

    Science.gov (United States)

    Springer, Armin; Kang, ChulHee; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Christiane; Pollmann, Stephan; Reinbothe, Steffen

    2016-03-22

    Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall.

  8. Mechanisms of Protein Synthesis in Chloroplasts: How to Design Translatable mRNAs in Chloroplasts

    Institute of Scientific and Technical Information of China (English)

    M. Sugiura

    2007-01-01

    @@ Chloroplast transformation provides a powerful tool to produce useful proteins in plants. After completion of the chloroplast genome sequencing from tobacco plants (Shinozaki et al., 1986, Yukawa et al., 2005), Pal Maliga group developed the high-frequency chloroplast transformation system in tobacco (Svab and Maliga, 1993).

  9. Isolation of Chloroplasts from Plant Protoplasts.

    Science.gov (United States)

    Lung, Shiu-Cheung; Smith, Matthew D; Chuong, Simon D X

    2015-10-01

    Chloroplasts can be isolated from higher plants directly following homogenization; however, the resulting yield, purity, and intactness are often low, necessitating a large amount of starting material. This protocol is optimized to produce a high yield of pure chloroplasts from isolated Arabidopsis protoplasts. The two-part method is a simple, scaled-down, and low-cost procedure that readily provides healthy mesophyll protoplasts, which are then ruptured to release intact chloroplasts. Chloroplasts isolated using this method are competent for use in biochemical, cellular, and molecular analyses.

  10. 盐碱胁迫对尼罗罗非鱼鳃Na+/3HCO共转运子、碳酸酐酶基因表达的影响%Effects of salinity and alkalinity on mRNA expression of Na+/3HCO cotransporter and carbonic anhydrase genes fromOreochromis niloticus

    Institute of Scientific and Technical Information of China (English)

    梁从飞; 赵金良; 甘远迪; 王飞; Thammaratsuntorn Jeerawat; 伍勇; 李传阳; 罗明坤

    2016-01-01

    To understand fish osmotic adjustment mechanisms in saline and alkaline water, the partial cDNA se-quence was obtained from gills ofOreochromis niloticus. Physiological changes in serum osmolality, ion concen-tration (Na+, K+, Cl– and Ca2+), and gill carbonic anhydrase (CA) activities were determined, andCAandNBCe1 mRNA gene expressions under saline (10 g/L, 15 g/L NaCl), alkaline (1.5 g/L and 3 g/L NaHCO3), and sa-line-alkaline (salinity 10, 15 g/L NaCl; salinity 1.5, 3 g/L NaHCO3) conditions at different times (0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h) were compared. The results showed that serum osmolality, ion concentration, gill CA activity, CA andNBCe1 mRNA gene expression correlated positively with the strength of saline, alkaline and sa-line-alkaline stress. Over time, serum osmolality and ion concentration trends increased and then decreased. Os-motic pressure insaline and saline-alkaline water was higher than that in alkaline water. Gill CA activity in alkaline and saline-alkaline water was higher than that in saline water. Under low concentrations of stressors, CA activity reached its highest level at a later time. Slightly higherNBCe1 gene mRNA expression was detected in gills under high concentrations of stressors (P>0.05). GillCA mRNA expression in saline, alkaline and saline-alkaline water was increased, but the increase was more evident in alkaline and saline-alkaline water (P0.05)。单碱组和盐碱混合组鳃CA活性较单盐组高,低盐碱胁迫(盐度10,碱度1.5 g/L)下CA活性较晚达最高值;不同胁迫条件下, CA基因mRNA表达均表现上调,单碱、盐碱混合组更为显著(P<0.05),推测CA较NBCe1对体内3HCO-转运作用更为显著。研究结果为尼罗罗非鱼盐碱适应生理调节提供了基础资料。

  11. Rapid mass movement of chloroplasts during segment formation of the calcifying siphonalean green alga, Halimeda macroloba.

    Directory of Open Access Journals (Sweden)

    Anthony W D Larkum

    Full Text Available BACKGROUND: The calcifying siphonalean green alga, Halimeda macroloba is abundant on coral reefs and is important in the production of calcium carbonate sediments. The process by which new green segments are formed over-night is revealed here for the first time. METHODOLOGY/PRINCIPAL FINDINGS: Growth of new segments was visualised by epifluorescence and confocal microscopy and by pulse amplitude modulation (PAM fluorimetry. Apical colourless proto-segments were initiated on day 1, and formed a loose network of non-calcified, non-septate filaments, containing no chloroplasts. Rapid greening was initiated at dusk by i the mass movement of chloroplasts into these filaments from the parent segment and ii the growth of new filaments containing chloroplasts. Greening was usually complete in 3-5 h and certainly before dawn on day 2 when the first signs of calcification were apparent. Mass chloroplast movement took place at a rate of ∼0.65 µm/s. Photosynthetic yield and rate remained low for a period of 1 to several hours, indicating that the chloroplasts were made de novo. Use of the inhibitors colchicine and cytochalasin d indicated that the movement process is dependent on both microtubules and microfilaments. SIGNIFICANCE: This unusual process involves the mass movement of chloroplasts at a high rate into new segments during the night and rapid calcification on the following day and may be an adaptation to minimise the impact of herbivorous activity.

  12. Effects of denervation on expression of carbonic anhydrase Ⅲ and its phosphatase activity in skeletal muscle of rats%去神经对大鼠骨骼肌碳酸酐酶Ⅲ表达和磷酸酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    黄河; 任惠民

    2011-01-01

    目的 观察去神经对骨骼肌碳酸酐酶Ⅲ(carbonic anhydrase Ⅲ,CAⅢ)表达及其磷酸酶(phosphatase)活性的影响,探讨神经冲动受阻是否为重症肌无力(myasthenia gravis,MG)骨骼肌CAⅢ减少的原因.方法 定向切断支配大鼠趾长伸肌(extensor digitorum longus,EDL)和比目鱼肌(soleus,Sol)的神经纤维,术后第7、14、28和56天用Western blot分析EDL和Sol的CAⅢ水平,用固相膜上原位酶活性染色方法评估CAⅢ的磷酸酶活性.结果 (1)正常侧(即去神经对侧)Sol的CAⅢ水平远高于EDL,并且两者都表现出随时间增加(动物年龄增长)而增加的趋势.去神经后,EDL的CAⅢ水平随时间的延长而逐渐增加;Sol的CAⅢ水平则以14 d为分界先增加后降低.(2)正常侧Sol的CAⅢ的磷酸酶活性[随时间增加(动物年龄增长)呈逐渐增加的趋势]均高于EDL(变化不明显).去神经后,Sol的CAⅢ磷酸酶活性(第14、28、56天分别为14.39±1.93、11.48±1.46、9.04±1.46)明显低于正常侧(22.75±1.80、25.26±3.15、25.82±2.97,t=0.002、0.005、0.002,均P<0.05),EDL的CAⅢ磷酸酶活性与正常侧相比亦是降低,但差异无统计学意义.(3)正常侧EDL和Sol的CAⅢ蛋白表达水平和CAⅢ的磷酸酶活性相一致;去神经后CAⅢ蛋白表达水平和CAⅢ的磷酸酶活性发生了背离,即CAⅢ蛋白表达水平增加,但其磷酸酶活性却降低.结论 去神经所致的神经冲动传递障碍与MG自身抗体所致的神经冲动传递障碍对骨骼肌CAⅢ表达水平的影响不同,MG骨骼肌CAⅢ表达水平减少并非是其自身抗体所致的神经冲动传递障碍造成.%Objective To observe the effects of nerve impulses on the expression of carbonic anhydrase Ⅲ ( CAⅢ ) and its phosphatase activity, and to explore whether or not the cause of CAⅢ expressive decreased in skeletal muscles of myasthenia gravis( MG) is resulted from the obstruction of nerve impulse.Methods The motor nerves of extensor digitorum

  13. On the structure of the spinach chloroplast

    NARCIS (Netherlands)

    Thomas, J.B.; Bustraan, M.; Paris, C.H.

    1952-01-01

    The structure of spinach chloroplasts was investigated with the aid of the electron microscope. It has been established that: 1. 1. the outer membrane of the chloroplasts is composed of both proteins and lipoids. 2. 2. the stroma is also built up by these components. 3. 3. within the stroma memb

  14. Chloroplasts as functional organelles in animal tissues.

    Science.gov (United States)

    Trench, R K; Greene, R W; Bystrom, B G

    1969-08-01

    The marine gastropod molluscs Tridachia crispata, Tridachiella diomedea, and Placobranchus ianthobapsus (Sacoglossa, Opisthobranchia) possess free functional chloroplasts within the cells of the digestive diverticula, as determined by observations on ultrastructure, pigment analyses, and experiments on photosynthetic capacity. In the light, the chloroplasts incorporate H(14)CO(3) (-)in situ. Reduced radiocarbon is translocated to various chloroplast-free tissues in the animals. The slugs feed on siphonaceous algae from which the chloroplasts are derived. Pigments from the slugs and from known siphonaceous algae, when separated chromatographically and compared, showed similar components. Absorption spectra of extracts of slugs and algae were very similar. The larvae of the slugs are pigment-free up to the post-veliger stage, suggesting that chloroplasts are acquired de novo. with each new generation. PMID:5792329

  15. Generation of nitric oxide from nitrite by carbonic anhydrase

    DEFF Research Database (Denmark)

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B;

    2009-01-01

    bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced...

  16. Generation of nitric oxide from nitrite by carbonic anhydrase:

    DEFF Research Database (Denmark)

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank Bo;

    2009-01-01

    bicarbonate and nitrite, we hypothesized that CA uses nitrite as a substrate to produce the potent vasodilator nitric oxide (NO) to increase local blood flow to metabolically active tissues. Here we show that CA readily reacts with nitrite to generate NO, particularly at low pH, and that the NO produced...

  17. Crystal structure of Brinzolamide: a carbonic anhydrase inhibitor.

    Science.gov (United States)

    Zheng, Huirong; Lou, Benyong

    2016-05-01

    In crystal structure of the title compound, C12H21N3O5S3 [systematic name: (R)-4-ethyl-amino-2-(3-meth-oxy-prop-yl)-3,4-di-hydro-2H-thieno[3,2-e][1,2]thia-zine-6-sulfonamide 1,1-dioxide], there exist three kinds of hydrogen-bonding inter-actions. The sulfonamide group is involved in hydrogen bonding with the secondary amine and the meth-oxy O atom, resulting in the formation of layers parallel to the bc plane. The layers are linked by an N-H⋯O hydrogen bond involving a sulfonamide O atom as acceptor and the secondary amine H atom as donor, which gives rise to the formation of a unique bilayer structure. The absolute structure of the mol-ecule in the crystal was determined by resonant scattering [Flack parameter = 0.01 (4)]. PMID:27308020

  18. Crystal structure of Brinzolamide: a carbonic anhydrase inhibitor

    Directory of Open Access Journals (Sweden)

    Huirong Zheng

    2016-05-01

    Full Text Available In crystal structure of the title compound, C12H21N3O5S3 [systematic name: (R-4-ethylamino-2-(3-methoxypropyl-3,4-dihydro-2H-thieno[3,2-e][1,2]thiazine-6-sulfonamide 1,1-dioxide], there exist three kinds of hydrogen-bonding interactions. The sulfonamide group is involved in hydrogen bonding with the secondary amine and the methoxy O atom, resulting in the formation of layers parallel to the bc plane. The layers are linked by an N—H...O hydrogen bond involving a sulfonamide O atom as acceptor and the secondary amine H atom as donor, which gives rise to the formation of a unique bilayer structure. The absolute structure of the molecule in the crystal was determined by resonant scattering [Flack parameter = 0.01 (4].

  19. Crystal structure of Brinzolamide: a carbonic anhydrase inhibitor

    OpenAIRE

    Zheng, Huirong; Lou, Benyong

    2016-01-01

    In crystal structure of the title compound, C12H21N3O5S3 [systematic name: (R)-4-ethyl­amino-2-(3-meth­oxy­prop­yl)-3,4-di­hydro-2H-thieno[3,2-e][1,2]thia­zine-6-sulfonamide 1,1-dioxide], there exist three kinds of hydrogen-bonding inter­actions. The sulfonamide group is involved in hydrogen bonding with the secondary amine and the meth­oxy O atom, resulting in the formation of layers parallel to the bc plane. The layers are linked by an N—H⋯O hydrogen bond involving a sulfonamide O atom as a...

  20. Chloroplast protein targeting involves localized translation in Chlamydomonas

    OpenAIRE

    Uniacke, James; Zerges, William

    2009-01-01

    The compartmentalization of eukaryotic cells requires that newly synthesized proteins be targeted to the compartments in which they function. In chloroplasts, a few thousand proteins function in photosynthesis, expression of the chloroplast genome, and other processes. Most chloroplast proteins are synthesized in the cytoplasm, imported, and then targeted to a specific chloroplast compartment. The remainder are encoded by the chloroplast genome, synthesized within the organelle, and targeted ...

  1. Effects of solar UV radiation on photosynthesis and enzyme activities (carbonic anhydrase and nitrate reductase in marine macroalgae from southern Spain Efectos de la radiación solar UV sobre la fotosíntesis y actividades enzimáticas (anhidrasa carbónica y nitrato reductasa en macralgas marinas del sur de España

    Directory of Open Access Journals (Sweden)

    FÉLIX L. FIGUEROA

    2001-06-01

    Full Text Available The effects of solar ultraviolet (UV radiation during daily cycles on photosynthesis and two key enzymes involved in carbon incorporation, the carbonic anhydrase, and in inorganic nitrogen reduction, the nitrate reductase, of macroalgae from southern Spain are presented. During daily cycles, photoinhibition in several intertidal macroalgae, expressed as decrease in the effective quantum yield from the morning to noon time, was linearly dependent on the daily integrated irradiance. However, recovery, expressed as the increase in the effective quantum yield from noon to the afternoon, presented a different pattern; full recovery was found below daily integrated irradiance of 1.0 x10(4 kJ m-2. However, recovery reached only 50 % at higher irradiances. The existence of daily photoinhibition and full recovery in intertidal algae suggests that photoinhibition is a photoprotective mechanism against high solar radiation as in higher plants, and that patterns of photoinhibition and recovery are affected by accumulative doses. Activities of carbonic anhidrase and nitrate reductase were determined in three marine macroalgae (Plocamium cartilagineum, Ulva rigida and Fucus spiralis under full (PAR + UV-A + UV-B and excluded UV solar radiation (PAR. Under PAR + UV-A + UV-B, peaks of enzyme activity were found in P. cartilagineum during the evening, and accordingly to data previously published for other red macroalgae. This situation was modified by the absence of UV radiation since the increase in the activities was delayed several hours. In the three macroalgae and under full solar radiation, a significant and negative correlation was found only when data from nitrate reductase activity was shifted in time during at least four hours. This correlation is lost in Ulva rigida when UV radiation is excluded. The existence of these daily variations with a negative correlation of both enzyme activities could reflect a complex regulatory link between carbon and

  2. Inheritance of chloroplast DNA in Chlamydomonas reinhardtii

    OpenAIRE

    Grant, David M; Nicholas W. Gillham; Boynton, John E.

    1980-01-01

    Two symmetrically located deletions of approximately 100 base pairs each have been identified in chloroplast DNA of Chlamydomonas reinhardtii. Although present in a mutant strain that requires acetate for growth, both deletions have been shown to be distinct from the nonphotosynthetic phenotype of this strain. These physical markers in the chloroplast genome and maternally inherited genetic markers showed strict cotransmission in reciprocal crosses. Thus, our results are consistent with the l...

  3. Synthesis of medium-chain- length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering

    Institute of Scientific and Technical Information of China (English)

    WANG Yuhua; WU Zhongyi; ZHANG Xiuhai; CHEN Guoqiang; WU Qiong; HUANG Conglin; YANG Qing

    2005-01-01

    Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3′-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line S4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mcl- PHAs in tobacco via chloroplast genetic engineering.

  4. Evolution of the chloroplast division machinery

    Institute of Scientific and Technical Information of China (English)

    Hongbo GAO; Fuli GAO

    2011-01-01

    Chloroplasts are photosynthetic organelles derived from endosymbiotic cyanobacteria during evolution.Dramatic changes occurred during the process of the formation and evolution of chloroplasts,including the large-scale gene transfer from chloroplast to nucleus.However,there are still many essential characters remaining.For the chloroplast division machinery,FtsZ proteins,Ftn2,SulA and part of the division site positioning system- MinD and MinE are still conserved.New or at least partially new proteins,such as FtsZ family proteins FtsZl and ARC3,ARC6H,ARC5,PDV1,PDV2 and MCD1,were introduced for the division of chloroplasts during evolution.Some bacterial cell division proteins,such as FtsA,MreB,Ftn6,FtsW and Ftsl,probably lost their function or were gradually lost.Thus,the chloroplast division machinery is a dynamically evolving structure with both conservation and innovation.

  5. Complete chloroplast genome of Sedum sarmentosum and chloroplast genome evolution in Saxifragales.

    Directory of Open Access Journals (Sweden)

    Wenpan Dong

    Full Text Available Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC region, 16.670 bp of a small single-copy (SSC region, and a pair of 25,783 bp sequences of inverted repeats (IRs.The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.

  6. The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO{sub 2}-concentrating mechanisms in algae

    Energy Technology Data Exchange (ETDEWEB)

    Badger, M. R.; Andrews, T. J.; Whitney, S. M.; Ludwig, M.; Price, G. D. [Australian National Univ., Research School of Biological Sciences, Canberra, ACT (Australia); Yellowlees, D. C.; Leggat, W. [James Cook Univ., Dept of Biochemistry and Molecular Biology, Townsville, QLD (Australia)

    1998-06-01

    The potential diversity of Rubisco and chloroplast-based carbon dioxide concentrating mechanisms (CCMs) in green and non-green algae are examined. The review emphasized recent advances in understanding the subject and areas with future research potential. In general, the review found that Rubisco enzymes from algae have evolved a higher affinity for carbon dioxide when the algae have adopted a strategy for carbon dioxide fixation that does not utilize a CCM. This appears to be true for both the Green and Red Form I Rubisco enzymes found in green and non-green algae. In some microalgae there is a strong correlation between the existence of a high-affinity CCM physiology and the presence of pyrenoids, suggestive of the potential importance of these chloroplast Rubisco-containing bodies. In contrast, in macroalgae a greater diversity of the apparent relationships between pyrenoids and chloroplast features and the CCM physiology was found. With regard to future research, the function of the pyrenoid and other chloroplast features, the operation of chloroplast-based CCM, and the assessment of the coevolution of Rubisco, appeared to be the most promising areas. 109 refs., 4 tabs., 5 figs.

  7. Chloroplasts in seeds and dark-grown seedlings of lotus.

    Science.gov (United States)

    Ushimaru, Takashi; Hasegawa, Takahiro; Amano, Toyoki; Katayama, Masao; Tanaka, Shigeyasu; Tsuji, Hideo

    2003-03-01

    In most higher plants, mature dry seeds have no chloroplasts but etioplasts. Here we show that in a hydrophyte, lotus (Nelumbo nucifera), young chloroplasts already exist in shoots of mature dry seeds and that they give rise to mature chloroplasts during germination, even in darkness. These shoots contain chlorophyll and chlorophyll-binding proteins CP1 and LHCP. The unique features of chloroplast formation in N. nucifera suggest a unique adaptive strategy for seedling development correlated with the plant's habitat.

  8. A comparison of rice chloroplast genomes

    DEFF Research Database (Denmark)

    Tang, Jiabin; Xia, Hong'ai; Cao, Mengliang;

    2004-01-01

    Using high quality sequence reads extracted from our whole genome shotgun repository, we assembled two chloroplast genome sequences from two rice (Oryza sativa) varieties, one from 93-11 (a typical indica variety) and the other from PA64S (an indica-like variety with maternal origin of japonica......), which are both parental varieties of the super-hybrid rice, LYP9. Based on the patterns of high sequence coverage, we partitioned chloroplast sequence variations into two classes, intravarietal and intersubspecific polymorphisms. Intravarietal polymorphisms refer to variations within 93-11 or PA64S...... to intersubspecific polymorphisms. In our study, we found that the intersubspecific variations of 93-11 (indica) and PA64S (japonica) chloroplast genomes consisted of 72 single nucleotide polymorphisms and 27 insertions or deletions. The intersubspecific polymorphism rates between 93-11 and PA64S were 0...

  9. Genomics and chloroplast evolution: what did cyanobacteria do for plants?

    OpenAIRE

    Raven, J.A.; Allen, John

    2003-01-01

    The complete genome sequences of cyanobacteria and of the higher plant Arabidopsis thaliana leave no doubt that the plant chloroplast originated, through endosymbiosis, from a cyanobacterium. But the genomic legacy of cyanobacterial ancestry extends far beyond the chloroplast itself, and persists in organisms that have lost chloroplasts completely.

  10. Chloroplast signaling within, between and beyond cells.

    Directory of Open Access Journals (Sweden)

    Krzysztof eBobik

    2015-10-01

    Full Text Available The most conspicuous function of the plastid is oxygenic photosynthesis of chloroplasts, yet plastids are super-factories that produce a plethora of compounds that are indispensable for proper plant physiology and development. Given their origins as free-living prokaryotes, it is not surprising that the plastid possesses its own genome whose expression is essential to plastid function. This semi-autonomous character of plastids requires the existence of sophisticated regulatory mechanisms that provide reliable communication between them and other cellular compartments. Such intracellular signaling is necessary for coordinating whole-cell responses to constantly varying environmental cues and cellular metabolic needs. This is achieved by plastids acting as receivers and transmitters of specific signals that coordinate expression of the nuclear and plastid genomes according to particular needs. In this review we will consider the so-called retrograde signaling occurring between plastids and nucleus, and between plastids and other organelles. Another important role of the plastid we will discuss is the involvement of plastid signaling in biotic and abiotic stress that, in addition to influencing retrograde signaling has direct effects on several cellular compartments including the cell wall. We will also review recent evidence pointing to an intriguing function of chloroplasts in regulating intercellular symplasmic transport. Finally, we consider an intriguing yet neglected aspect of plant biology, chloroplast signaling from the perspective of the entire plant. Thus, accumulating evidence highlights that chloroplasts, with their complex signaling pathways, provide a mechanism for exquisite regulation of plant development, metabolism and responses to the environment. As chloroplast processes are targeted for engineering for improved productivity the effect of such modifications on chloroplast signaling will have to be carefully considered in order

  11. Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

    OpenAIRE

    Marta Brozynska; Agnelo Furtado; Robert James Henry

    2014-01-01

    Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genom...

  12. Energetic cost of protein import across the envelope membranes of chloroplasts.

    Science.gov (United States)

    Shi, Lan-Xin; Theg, Steven M

    2013-01-15

    Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ~3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranes--only the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ~650 ATP molecules. This translates to a ΔG(protein) (transport) of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ~0.6% of the total light-saturated energy output of the organelle. PMID:23277572

  13. Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants

    OpenAIRE

    Michelet, Laure; Zaffagnini, Mirko; Marchand, Christophe; Collin, Valérie; Decottignies, Paulette; Tsan, Pascale; Lancelin, Jean-Marc; Trost, Paolo; Miginiac-Maslow, Myroslawa; Noctor, Graham; Lemaire, Stéphane D.

    2005-01-01

    Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No...

  14. Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase.

    OpenAIRE

    Chiadmi, M.; Navaza, A; Miginiac-Maslow, M; Jacquot, J P; Cherfils, J

    1999-01-01

    Sunlight provides the energy source for the assimilation of carbon dioxide by photosynthesis, but it also provides regulatory signals that switch on specific sets of enzymes involved in the alternation of light and dark metabolisms in chloroplasts. Capture of photons by chlorophyll pigments triggers redox cascades that ultimately activate target enzymes via the reduction of regulatory disulfide bridges by thioredoxins. Here we report the structure of the oxidized, low-activity form of chlorop...

  15. Alkyl sulfonic acide hydrazides: Synthesis, characterization, computational studies and anticancer, antibacterial, anticarbonic anhydrase II (hCA II) activities

    Science.gov (United States)

    O. Ozdemir, Ummuhan; İlbiz, Firdevs; Balaban Gunduzalp, Ayla; Ozbek, Neslihan; Karagoz Genç, Zuhal; Hamurcu, Fatma; Tekin, Suat

    2015-11-01

    Methane sulfonic acide hydrazide, CH3SO2NHNH2 (1), ethane sulfonic acide hydrazide, CH3CH2SO2NHNH2 (2), propane sulfonic acide hydrazide, CH3CH2CH2SO2NHNH2 (3) and butane sulfonic acide hydrazide, CH3CH2CH2CH2SO2NHNH2 (4) have been synthesized as homologous series and characterized by using elemental analysis, spectrophotometric methods (1H-13C NMR, FT-IR, LC-MS). In order to gain insight into the structure of the compounds, we have performed computational studies by using 6-311G(d, p) functional in which B3LYP functional were implemented. The geometry of the sulfonic acide hydrazides were optimized at the DFT method with Gaussian 09 program package. A conformational analysis of compounds were performed by using NMR theoretical calculations with DFT/B3LYP/6-311++G(2d, 2p) level of theory by applying the (GIAO) approach. The anticancer activities of these compounds on MCF-7 human breast cancer cell line investigated by comparing IC50 values. The antibacterial activities of synthesized compounds were studied against Gram positive bacteria; Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Bacillus cereus NRRL-B-3711, Enterococcus faecalis ATCC 29212 and Gram negative bacteria; Escherichia coli ATCC 11230, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumonia ATCC 70063 by using the disc diffusion method. The inhibition activities of these compounds on carbonic anhydrase II enzyme (hCA II) have been investigated by comparing IC50 and Ki values. The biological activity screening shows that butane sulfonic acide hydrazide (4) has more activity than the others against tested breast cancer cell lines MCF-7, Gram negative/Gram positive bacteria and carbonic anhydrase II (hCA II) isoenzyme.

  16. A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiaozhen Huang; Xiaoyan Zhang; Shuhua Yang

    2009-01-01

    To understand the molecular mechanisms underlying chloroplast development, we isolated and characterized the albino mutant emb1303-1 in Arabidopsis. The mutant displayed a severe dwarf phenotype with small albino rosette leaves and short roots on a synthetic medium containing sucrose. It is pigment-deficient and seedling lethal when grown in soil. Embryo development was delayed in the mutant, although seed germination was not significantly im-paired. The plastids of emb1303-1 were arrested in early developmental stages without the classical stack of thylakoid membrane. Genetic and molecular analyses uncovered that the EMB1303 gene encodes a novel chloroplast-localized protein. Mieroarray and RT-PCR analyses revealed that a number of nuclear-and plastid-encoded genes involved in photosynthesis and chloroplast biogenesis were substantially downregulated in the mutant. Moreover, the accu-mulation of several major chloroplast proteins was severely compromised in emb1303-1. These results suggest that EMBI303 is essential for chloroplast development.

  17. Direct chloroplast sequencing: comparison of sequencing platforms and analysis tools for whole chloroplast barcoding.

    Directory of Open Access Journals (Sweden)

    Marta Brozynska

    Full Text Available Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina and Ion Torrent (Life Technology sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare. Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.

  18. Origin of a chloroplast protein importer

    OpenAIRE

    Bölter, Bettina; Soll, Jürgen; Schulz, Alexander; Hinnah, Silke; Wagner, Richard

    1998-01-01

    During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import s...

  19. Expressing PHB synthetic genes through chloroplast genetic engineering

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Chloroplast integration and expression vector containing expression cassettes for phbB, phbA, phbC and aadA genes was constructed and bombarded into the tobacco chloroplast genome. Transplastomic plants were analyzed with PCR and Southern blot. Their homoplastomy was also judged. Northern dot and RT-PCR analysis were employed to investigate transgene expression at transcriptional level. The results indicate that the chloroplast transformation system is compatible for poly-3-hydroxybutyrate (PHB) production.

  20. Nanophotonics of Chloroplasts for Bio-Inspired Solar Energy Materials

    Science.gov (United States)

    Gourley, Paul L.; Gourley, Cheryl R.

    2011-03-01

    In the search for new energy sources, lessons can be learned from chloroplast photonics. The nano-architecture of chloroplasts is remarkably well-adapted to mediate sunlight interactions for efficient energy conversion. We carried out experiments with chloroplasts isolated from spinach and leaf lettuce to elucidate the relationship between nano-architecture, biomolecular composition and photonic properties. We obtained high-resolution microscopic images of single chloroplasts to identify geometries of chloroplasts and interior grana. We performed micro-spectroscopy to identify strengths of absorption and fluorescence transitions and related them to broadband reflectance and transmittance spectra of whole leaf structures. Finally, the nonlinear optical properties were investigated with nanolaser spectroscopy by placing chloroplasts into micro-resonators and optically pumping. These spectra reveal chloroplast photonic modes and allow measurement of single chloroplast light scattering cross section, polarizability, and refractive index. The nanolaser spectra recorded at increasing pump powers enabled us to observe non-linear optics, photon dynamics, and stimulated emission from single chloroplasts. All of these experiments provide insight into plant photonics and inspiration of paradigms for synthetic biomaterials to harness sunlight in new ways.

  1. Complex chloroplast RNA metabolism: just debugging the genetic programme?

    Directory of Open Access Journals (Sweden)

    Schmitz-Linneweber Christian

    2008-08-01

    Full Text Available Abstract Background The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity. Results We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants. Conclusion Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.

  2. Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis.

    Science.gov (United States)

    Oikawa, Kazusato; Matsunaga, Shigeru; Mano, Shoji; Kondo, Maki; Yamada, Kenji; Hayashi, Makoto; Kagawa, Takatoshi; Kadota, Akeo; Sakamoto, Wataru; Higashi, Shoichi; Watanabe, Masakatsu; Mitsui, Toshiaki; Shigemasa, Akinori; Iino, Takanori; Hosokawa, Yoichiroh; Nishimura, Mikio

    2015-03-30

    Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

  3. Photosynthetic 14C fixation in leaves and isolated chloroplasts of some scrub species under the influence of paraquat and 2,4,5-T

    International Nuclear Information System (INIS)

    The diurnal course of carbon dioxide uptake in the leaves of six nonsucculent semiarid scrub species was investigated. Five nonsucculent scrub species, Carissa spinarum, Maba buxifolia, Flacourtia sepiaria, Gymnosporia emerginata and Dodonaea viscosa exhibited nocturnal carbon assimilation while day time CO2 uptake was predominant in Chomelia asiatica. Foliar application of paraquat (100 mg 1sup(-1)) or 2,4,5-T (2000 mg 1sup(-1)) caused a marked inhibition in the dark uptake of CO2. On the other hand, carbon uptake during day was slightly reduced. Studies on the carbon assimilation of isolated chloroplasts under the influence of paraquat or 2,4,5-T revealed that paraquat is more effective than 2,4,5-T in the suppression of carbon assimilation of in vivo and in vitro chloroplasts. The data suggested that the process of nocturnal carbon assimilation is far more sensitive to paraquat than the light dependent carbon fixation during day. (author)

  4. Photochemical properties of mesophyll and bundle sheath chloroplasts of maize.

    Science.gov (United States)

    Bazzaz, M B; Govindjee

    1973-09-01

    Several photochemical and spectral properties of maize (Zea mays) bundle sheath and mesophyll chloroplasts are reported that provide a better understanding of the photosynthetic apparatus of C(4) plants. The difference absorption spectrum at 298 K and the fluorescence excitation and emission spectra of chlorophyll at 298 K and 77 K provide new information on the different forms of chlorophyll a in bundle sheath and mesophyll chloroplasts: the former contain, relative to short wavelength chlorophyll a forms, more long wavelength chlorophyll a form (e.g. chlorophyll a 693 and chlorophyll a 705) and less chlorophyll b than the latter. The degree of polarization of chlorophyll a fluorescence is 6% in bundle sheath and 4% in mesophyll chloroplasts. This result is consistent with the presence of relatively high amounts of oriented long wavelength forms of chlorophyll a in bundle sheath compared to mesophyll chloroplasts. The relative yield of variable, with respect to constant, chorophyll a fluorescence in mesophyll chloroplasts is more than twice that in bundle sheath chloroplast. Furthermore, the relative yield of total chlorophyll a fluorescence is 40% lower in bundle sheath compared to that in mesophyll chloroplasts. This is in agreement with the presence of the higher ratio of the weakly fluorescent pigment system I to pigment system II in bundle sheath than in mesophyll chloroplast. The efficiency of energy transfer from chlorophyll b and carotenoids to chlorophyll a are calculated to be 100 and 50%, respectively, in both types of chloroplasts. Fluorescence quenching of atebrin, reflecting high energy state of chloroplasts, is 10 times higher in mesophyll chloroplasts than in bundle sheath chloroplasts during noncyclic electron flow but is equal during cyclic flow. The entire electron transport chain is shown to be present in both types of chloroplasts, as inferred from the antagonistic effect of red (650 nm) and far red (710 nm) lights on the absorbance changes at

  5. Orientation of the pigment molecules in the chloroplast

    NARCIS (Netherlands)

    Goedheer, J.C.

    1955-01-01

    Dichroism, absorption anisotropy, and anomal dispersion of birefringence were measured in the big lamellate chloroplasts of Mougeotia. The results of these measurements indicate a certain orientation of the chlorophyll molecules, and to a smaller extent, of the carotenoids in the chloroplast. In sp

  6. Tools for regulated gene expression in the chloroplast of Chlamydomonas.

    Science.gov (United States)

    Rochaix, Jean-David; Surzycki, Raymond; Ramundo, Silvia

    2014-01-01

    The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun, and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter drives the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5'-untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or transgene by placing it under the control of the psbD 5'-untranslated region. PMID:24599871

  7. Origin and evolution of the chloroplast division machinery.

    Science.gov (United States)

    Miyagishima, Shin-Ya

    2005-10-01

    Chloroplasts were originally established in eukaryotes by the endosymbiosis of a cyanobacterium; they then spread through diversification of the eukaryotic hosts and subsequent engulfment of eukaryotic algae by previously nonphotosynthetic eukaryotes. The continuity of chloroplasts is maintained by division of preexisting chloroplasts. Like their ancestors, chloroplasts use a bacterial division system based on the FtsZ ring and some associated factors, all of which are now encoded in the host nuclear genome. The majority of bacterial division factors are absent from chloroplasts and several new factors have been added by the eukaryotic host. For example, the ftsZ gene has been duplicated and modified, plastid-dividing (PD) rings were most likely added by the eukaryotic host, and a member of the dynamin family of proteins evolved to regulate chloroplast division. The identification of several additional proteins involved in the division process, along with data from diverse lineages of organisms, our current knowledge of mitochondrial division, and the mining of genomic sequence data have enabled us to begin to understand the universality and evolution of the division system. The principal features of the chloroplast division system thus far identified are conserved across several lineages, including those with secondary chloroplasts, and may reflect primeval features of mitochondrial division. PMID:16143878

  8. Expression of eukaryotic polypeptides in chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P.

    2013-06-04

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  9. Chloroplast division during leaf development of Xanthium pensylvanicum Wallr. (Compositae

    Directory of Open Access Journals (Sweden)

    Roman Maksymowych

    2014-02-01

    Full Text Available Division and growth of chloroplasts was studied during leaf development of Xanthium pensylvanicum at various stages of development represented by the leaf plastochron index.Between leaf plastochron indices -1.00 and 2.56 chloroplast division was observed with little enlargement. Between 2.50 and 5.00 chloroplasts enlarged in diameter with an average rate of 0.21 µm per day. At leaf plastochron index 5.00 chloroplasts attained their mature size of 6.12 µm. No chloroplast division was found after leaf plastochron index 2.50. A change in shape of plastids from spherical proplastids to discoidal accompanied their growth during stages 2.50 and 5.00.

  10. Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum (Bacillariophyta)

    Institute of Scientific and Technical Information of China (English)

    Xiao-Juan Liu; Shun-Shan Duan; Ai-Fen Li; Kai-Feng Sun

    2009-01-01

    Responses of the photosynthetic activity of Phaeodactylum tricornutum (Bacillariophyta) to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem (PS) Ⅰ and PS Ⅱ and the values of F6851F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.

  11. Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

    OpenAIRE

    Puntarulo, Susana; Jasid, Sebastián; Simontacchi, Marcela

    2007-01-01

    Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique.1 Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min−1 mg−1 protein in the presence of 1 mM NaNO2. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min−1 mg−1...

  12. Origins of prokaryotes, eukaryotes, mitochondria, and chloroplasts

    Science.gov (United States)

    Schwartz, R. M.; Dayhoff, M. O.

    1978-01-01

    A computer branching model is used to analyze cellular evolution. Attention is given to certain key amino acids and nucleotide residues (ferredoxin, 5s ribosomal RNA, and c-type cytochromes) because of their commonality over a wide variety of cell types. Each amino acid or nucleotide residue is a sequence in an inherited biological trait; and the branching method is employed to align sequences so that changes reflect substitution of one residue for another. Based on the computer analysis, the symbiotic theory of cellular evolution is considered the most probable. This theory holds that organelles, e.g., mitochondria and chloroplasts invaded larger bodies, e.g., bacteria, and combined functions to form eucaryotic cells.

  13. Chloroplast genome variation in upland and lowland switchgrass

    Science.gov (United States)

    Switchgrass (Panicum virgatum L.) exists at multiple ploidies and two phenotypically distinct ecotypes. To facilitate interploidal comparisons and to understand the extent of sequence variation within existing breeding pools, two complete switchgrass chloroplast genomes were sequenced from individu...

  14. New Insights into Dynamic Actin-Based Chloroplast Photorelocation Movement

    Institute of Scientific and Technical Information of China (English)

    Sam-Geun Kong; Masamitsu Wada

    2011-01-01

    Chloroplast movement is essential for plants to survive under various environmental light conditions.Phototropins-plant-specific blue-light-activated receptor kinases-mediate the response by perceiving light intensity and direction.Recently,novel chloroplast actin (cp-actin) filaments have been identified as playing a pivotal role in the directional chloroplast photorelocation movement.Encouraging progress has recently been made in this field of research through molecular genetics and cell biological analyses.This review describes factors that have been identified as being involved in chloroplast movement and their roles in the regulation of cp-actin filaments,thus providing a basis for reflection on their biochemical activities and functions.

  15. Separation of Chloroplast Pigments Using Reverse Phase Chromatography.

    Science.gov (United States)

    Reese, R. Neil

    1997-01-01

    Presents a protocol that uses reverse phase chromatography for the separation of chloroplast pigments. Provides a simple and relatively safe procedure for use in teaching laboratories. Discusses pigment extraction, chromatography, results, and advantages of the process. (JRH)

  16. Full transcription of the chloroplast genome in photosynthetic eukaryotes.

    Science.gov (United States)

    Shi, Chao; Wang, Shuo; Xia, En-Hua; Jiang, Jian-Jun; Zeng, Fan-Chun; Gao, Li-Zhi

    2016-01-01

    Prokaryotes possess a simple genome transcription system that is different from that of eukaryotes. In chloroplasts (plastids), it is believed that the prokaryotic gene transcription features govern genome transcription. However, the polycistronic operon transcription model cannot account for all the chloroplast genome (plastome) transcription products at whole-genome level, especially regarding various RNA isoforms. By systematically analyzing transcriptomes of plastids of algae and higher plants, and cyanobacteria, we find that the entire plastome is transcribed in photosynthetic green plants, and that this pattern originated from prokaryotic cyanobacteria - ancestor of the chloroplast genomes that diverged about 1 billion years ago. We propose a multiple arrangement transcription model that multiple transcription initiations and terminations combine haphazardly to accomplish the genome transcription followed by subsequent RNA processing events, which explains the full chloroplast genome transcription phenomenon and numerous functional and/or aberrant pre-RNAs. Our findings indicate a complex prokaryotic genome regulation when processing primary transcripts. PMID:27456469

  17. Transport of Ions Across the Inner Envelope Membrane of Chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    McCarty, R. E.

    2004-06-02

    The technical report outlines the results of nine years of research on how ions cross the inner envelope membrane of chloroplasts. The ions include protons, nitrite, calcium and ferrous iron. Bicarbonate transport was also studied.

  18. The complete chloroplast genome sequence of Zanthoxylum piperitum.

    Science.gov (United States)

    Lee, Jonghoon; Lee, Hyeon Ju; Kim, Kyunghee; Lee, Sang-Choon; Sung, Sang Hyun; Yang, Tae-Jin

    2016-09-01

    The complete chloroplast genome sequence of Zanthoxylum piperitum, a plant species with useful aromatic oils in family Rutaceae, was generated in this study by de novo assembly with whole-genome sequence data. The chloroplast genome was 158 154 bp in length with a typical quadripartite structure containing a pair of inverted repeats of 27 644 bp, separated by large single copy and small single copy of 85 340 bp and 17 526 bp, respectively. The chloroplast genome harbored 112 genes consisting of 78 protein-coding genes 30 tRNA genes and 4 rRNA genes. Phylogenetic analysis of the complete chloroplast genome sequences with those of known relatives revealed that Z. piperitum is most closely related to the Citrus species. PMID:26260183

  19. A Nucleus-Encoded Chloroplast Protein YL1 Is Involved in Chloroplast Development and Efficient Biogenesis of Chloroplast ATP Synthase in Rice

    Science.gov (United States)

    Chen, Fei; Dong, Guojun; Wu, Limin; Wang, Fang; Yang, Xingzheng; Ma, Xiaohui; Wang, Haili; Wu, Jiahuan; Zhang, Yanli; Wang, Huizhong; Qian, Qian; Yu, Yanchun

    2016-09-01

    Chloroplast ATP synthase (cpATPase) is an importance thylakoid membrane-associated photosynthetic complex involved in the light-dependent reactions of photosynthesis. In this study, we isolated and characterized a rice (Oryza sativa) mutant yellow leaf 1 (yl1), which exhibits chlorotic leaves throughout developmental stages. The YL1 mutation showed reduced chlorophyll contents, abnormal chloroplast morphology, and decreased photochemical efficiency. Moreover, YL1 deficiency disrupts the expression of genes associated with chloroplast development and photosynthesis. Molecular and genetic analyses revealed that YL1 is a nucleus-encoded protein with a predicted transmembrane domain in its carboxyl-terminus that is conserved in the higher plant kingdom. YL1 localizes to chloroplasts and is preferentially expressed in green tissues containing chloroplasts. Immunoblot analyses showed that inactivation of YL1 leads to drastically reduced accumulation of AtpA (α) and AtpB (β), two core subunits of CF1αβ subcomplex of cpATPase, meanwhile, a severe decrease (ca. 41.7%) in cpATPase activity was observed in the yl1-1 mutant compared with the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays revealed a specific interaction between YL1 and AtpB subunit of cpATPase. Taken together, our results suggest that YL1 is a plant lineage-specific auxiliary factor involved in the biogenesis of the cpATPase complex, possibly via interacting with the β-subunit.

  20. Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome.

    Directory of Open Access Journals (Sweden)

    Alison Gonçalves Nazareno

    Full Text Available Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq. L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp and a small single copy region (SSC, 17,586 bp separated by inverted repeat regions (IRs, 25,789 bp. In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering

  1. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. PMID:25554634

  2. Sequence evidence for the symbiotic origins of chloroplasts and mitochondria

    Science.gov (United States)

    George, D. G.; Hunt, L. T.; Dayhoff, M. O.

    1983-01-01

    The origin of mitochondria and chloroplasts is investigated on the basis of prokaryotic and early-eukaryotic evolutionary trees derived from protein and nucleic-acid sequences by the method of Dayhoff (1979). Trees for bacterial ferrodoxins, 5S ribosomal RNA, c-type cytochromes, the lipid-binding subunit of ATPase, and dihydrofolate reductase are presented and discussed. Good agreement among the trees is found, and it is argued that the mitochondria and chloroplasts evolved by multiple symbiotic events.

  3. Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

    Institute of Scientific and Technical Information of China (English)

    YANG Zongqi; LI yinü; CHEN Feng; LI Dong; ZHANG Zhifang; LIU Yanxin; ZHENG Dexian; WANG Yong; SHEN Guifang

    2006-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selectively apoptosis in various tumor cells and virus-infected cells, but rarely in normal cells. A chloroplast expression vector, p64TRAIL, containing the cDNA coding for the soluble TRAIL (sTRAIL), was constructed with clpP-trnL-petB-chlL-rpl23-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spectinomycin-resistant aadA gene as a select marker. The plasmid p64TRAIL was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Three independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the sTRAIL coding region DNA and cultivation cells in the dark all showed that the exogenous DNA had been integrated into chloroplast genome of C. reinhardtii. Western blot analysis showed that human soluble TRAIL was expressed in C. reinhardtii chloroplast. The densitometric analysis of Western blot indicated that the expressed human sTRAIL protein in the chloroplasts of C. reinhardtii accounted for about 0.43%-0.67% of the total soluble proteins.These experimental results demonstrated the possibility of using transgenic chloroplasts of green alga as bioreactors for production of biopharmaceuticals.

  4. A carboxysomal carbon-concentrating mechanism in the cyanelles of the 'coelacanth' of the algal world, Cyanophora paradoxa?

    Science.gov (United States)

    Fathinejad, Sara; Steiner, Jürgen M; Reipert, Siegfried; Marchetti, Martina; Allmaier, Günter; Burey, Suzanne C; Ohnishi, Norikazu; Fukuzawa, Hideya; Löffelhardt, Wolfgang; Bohnert, Hans J

    2008-05-01

    Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.

  5. Multiple feedbacks between chloroplast and whole plant in the context of plant adaptation and acclimation to the environment

    Science.gov (United States)

    Demmig-Adams, Barbara; Stewart, Jared J.; Adams, William W.

    2014-01-01

    This review focuses on feedback pathways that serve to match plant energy acquisition with plant energy utilization, and thereby aid in the optimization of chloroplast and whole-plant function in a given environment. First, the role of source–sink signalling in adjusting photosynthetic capacity (light harvesting, photochemistry and carbon fixation) to meet whole-plant carbohydrate demand is briefly reviewed. Contrasting overall outcomes, i.e. increased plant growth versus plant growth arrest, are described and related to respective contrasting environments that either do or do not present opportunities for plant growth. Next, new insights into chloroplast-generated oxidative signals, and their modulation by specific components of the chloroplast's photoprotective network, are reviewed with respect to their ability to block foliar phloem-loading complexes, and, thereby, affect both plant growth and plant biotic defences. Lastly, carbon export capacity is described as a newly identified tuning point that has been subjected to the evolution of differential responses in plant varieties (ecotypes) and species from different geographical origins with contrasting environmental challenges. PMID:24591724

  6. Photosynthetic assimilation of 14C in isolated chloroplasts in the presence of NO3-, SO4- and NH4+

    International Nuclear Information System (INIS)

    Quantitative changes in carbon photosynthesis assimilation occurring as an effect of varying nitrate, sulfate and ammonia ions in the incubation medium were studied in isolated chloroplasts of spinach. Carbon photosynthetic assimilation is enhanced under the influence of rising nitrate anion concentrations to a certain level. The percentage of 14C concent in the insoluble products is also raised while in glycolic acid it is reduced. The nitrate anion has an effect similar to that of the bicarbonic anion the same processes. Ammonium and sulfate ions have the opposite effect. It can be assumed that the established effect of the ions studied is due to the influence they have on photosynthetic phosphorylation. (author)

  7. The puzzle of chloroplast vesicle transport – involvement of GTPases

    Directory of Open Access Journals (Sweden)

    Sazzad eKarim

    2014-09-01

    Full Text Available In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum (ER network, Golgi bodies, secretory granules, endosome and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence indicates presence of a vesicle transport system in chloroplasts. Little is known about the protein components of this system. However, as chloroplasts harbour the photosynthetic apparatus that ultimately supports most organisms on the planet, close attention to their pathways is warranted. This may also reveal novel diversification and/or distinct solutions to the problems posed by the targeted intra-cellular trafficking of important molecules. To date two homologues to well-known yeast cytosolic vesicle transport proteins, CPSAR1 and CPRabA5e, have been shown to have roles in chloroplast vesicle transport, both being GTPases. Bioinformatic data indicate that several homologues of cytosolic vesicle transport system components are putatively chloroplast-localized and in addition other proteins have been implicated to participate in chloroplast vesicle transport, including vesicle-inducing protein in plastids 1 (VIPP1, thylakoid formation 1 (THF1, snowy cotyledon 2/cotyledon chloroplast biogenesis factor (SCO2/CYO1, curvature thylakoid 1 (CURT1 proteins, and a dynamin like GTPase FZO-like (FZL protein. Several putative potential cargo proteins have also been identified, including building blocks of the photosynthetic apparatus. Here we discuss details of the largely unknown putative chloroplast vesicle transport system, focusing on GTPase-related components.

  8. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Directory of Open Access Journals (Sweden)

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  9. Chloroplast genome structure in Ilex (Aquifoliaceae).

    Science.gov (United States)

    Yao, Xin; Tan, Yun-Hong; Liu, Ying-Ying; Song, Yu; Yang, Jun-Bo; Corlett, Richard T

    2016-01-01

    Aquifoliaceae is the largest family in the campanulid order Aquifoliales. It consists of a single genus, Ilex, the hollies, which is the largest woody dioecious genus in the angiosperms. Most species are in East Asia or South America. The taxonomy and evolutionary history remain unclear due to the lack of a robust species-level phylogeny. We produced the first complete chloroplast genomes in this family, including seven Ilex species, by Illumina sequencing of long-range PCR products and subsequent reference-guided de novo assembly. These genomes have a typical bicyclic structure with a conserved genome arrangement and moderate divergence. The total length is 157,741 bp and there is one large single-copy region (LSC) with 87,109 bp, one small single-copy with 18,436 bp, and a pair of inverted repeat regions (IR) with 52,196 bp. A total of 144 genes were identified, including 96 protein-coding genes, 40 tRNA and 8 rRNA. Thirty-four repetitive sequences were identified in Ilex pubescens, with lengths >14 bp and identity >90%, and 11 divergence hotspot regions that could be targeted for phylogenetic markers. This study will contribute to improved resolution of deep branches of the Ilex phylogeny and facilitate identification of Ilex species. PMID:27378489

  10. Comparison of intraspecific, interspecific and intergeneric chloroplast diversity in Cycads.

    Science.gov (United States)

    Jiang, Guo-Feng; Hinsinger, Damien Daniel; Strijk, Joeri Sergej

    2016-01-01

    Cycads are among the most threatened plant species. Increasing the availability of genomic information by adding whole chloroplast data is a fundamental step in supporting phylogenetic studies and conservation efforts. Here, we assemble a dataset encompassing three taxonomic levels in cycads, including ten genera, three species in the genus Cycas and two individuals of C. debaoensis. Repeated sequences, SSRs and variations of the chloroplast were analyzed at the intraspecific, interspecific and intergeneric scale, and using our sequence data, we reconstruct a phylogenomic tree for cycads. The chloroplast was 162,094 bp in length, with 133 genes annotated, including 87 protein-coding, 37 tRNA and 8 rRNA genes. We found 7 repeated sequences and 39 SSRs. Seven loci showed promising levels of variations for application in DNA-barcoding. The chloroplast phylogeny confirmed the division of Cycadales in two suborders, each of them being monophyletic, revealing a contradiction with the current family circumscription and its evolution. Finally, 10 intraspecific SNPs were found. Our results showed that despite the extremely restricted distribution range of C. debaoensis, using complete chloroplast data is useful not only in intraspecific studies, but also to improve our understanding of cycad evolution and in defining conservation strategies for this emblematic group. PMID:27558458

  11. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    Science.gov (United States)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  12. Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Chloroplastic respiration was monitored by measuring 14CO2 from 14C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast, The patterns of 14CO2 evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolypyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The Km for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of 14CO2 was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO2 evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO2 evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH4Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolypyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to Co2 and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism

  13. Chloroplast DNA Diversity of Oak Species in Eastern Romania

    Directory of Open Access Journals (Sweden)

    Ioan Calin MOLDOVAN

    2010-12-01

    Full Text Available The chloroplast DNA of 34 sessile oak (Quercus petraea and 27 pedunculate oak (Q. robur populations covering the entire natural distribution of the two oak species in Eastern Romania was investigated using four large regions of the chloroplast genome by PCR and RFLP technique. A total of seven chloroplast DNA haplotypes sensu lato have been observed by analysing 305 mature trees. However, due to the high resolution of the electrophoresis method a total of 22 chloroplast variants could have been detected, with new mutations and fragment combinations in two of the amplified regions: psbC/trnD and trnT/trnF. All of the haplotypes belong to the phylogenetic lineages A and E, which originate from the Balkan Peninsula. Most of genetic diversity is distributed among populations (GST=0.779. The chloroplast DNA haplotypes are shared by the two oak species. Different dispersal abilities may explain the higher value of genetic differentiation among populations in sessile oak than in pedunculate oak.

  14. The complete chloroplast genome of Capsicum frutescens (Solanaceae)1

    Science.gov (United States)

    Shim, Donghwan; Raveendar, Sebastin; Lee, Jung-Ro; Lee, Gi-An; Ro, Na-Young; Jeon, Young-Ah; Cho, Gyu-Taek; Lee, Ho-Sun; Ma, Kyung-Ho; Chung, Jong-Wook

    2016-01-01

    Premise of the study: We report the complete sequence of the chloroplast genome of Capsicum frutescens (Solanaceae), a species of chili pepper. Methods and Results: Using an Illumina platform, we sequenced the chloroplast genome of C. frutescens. The total length of the genome is 156,817 bp, and the overall GC content is 37.7%. A pair of 25,792-bp inverted repeats is separated by small (17,853 bp) and large (87,380 bp) single-copy regions. The C. frutescens chloroplast genome encodes 132 unique genes, including 87 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Of these, seven genes are duplicated in the inverted repeats and 12 genes contain one or two introns. Comparative analysis with the reference chloroplast genome revealed 125 simple sequence repeat motifs and 34 variants, mostly located in the noncoding regions. Conclusions: The complete chloroplast genome sequence of C. frutescens reported here is a valuable genetic resource for Capsicum species. PMID:27213127

  15. Molecular biology and physiology of isolated chloroplasts from the algae Vaucheria

    OpenAIRE

    Didriksen, Alena

    2010-01-01

    Sea slugs of the genus Elysia (e.g. E. chlorotica) are known for their ability to incorporate chloroplasts from the yellow-green alga Vaucheria litorea. These “kleptoplasts” stay active in the digestive tract of the sea slug for several months. Chloroplasts from Vaucheria litorea are also reported to be significantly more stable after in vitro isolation than chloroplasts of other algae or of higher plants. In organello assays with isolated chloroplasts are used in studies on photosynthetical ...

  16. Chloroplast degeneration and its inhibition by kinetin in detached leaves of Cichorium intybus L.

    OpenAIRE

    F. Młodzianowski; L. Młodzanowska

    2015-01-01

    In the chicory (Cichorium intybus L. var. sativum cv. Polanowicka) leaves two types of chloroplasts are present differing by their degree of osmiophility of the thylakoid inside. This type of differentiation of chloroplasts has so far been found only in several plant species. The process of chloroplast degeneration in darkness is described. In osmiophilic chloroplasts at certain stage of degeneration minutely layered giant grana were found. Kinetin markedly inhibited the process of chloroplas...

  17. Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43 Is Required for Chloroplast Development and Photosynthesis.

    Directory of Open Access Journals (Sweden)

    Xiang-guang Lv

    Full Text Available A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS-induced IR64 (Oryza sativa L. ssp. indica mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43 with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43 was required for the normal development of chloroplasts and photosynthesis in rice.

  18. Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis.

    Science.gov (United States)

    Lv, Xiang-guang; Shi, Yong-feng; Xu, Xia; Wei, Yan-lin; Wang, Hui-mei; Zhang, Xiao-bo; Wu, Jian-li

    2015-01-01

    A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)-induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice.

  19. Factors affecting the stability of chloroplast membranes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Takaoki, T.; Torres-Pereira, J.; Packer, L.

    1974-01-01

    Factors which affect the stability of light-induced atebrin fluorescence quenching activity in chloroplast membranes, a measure of the electron transport dependent formation of energy-linked H/sup +/ gradients, were investigated in vitro. Class II spinach chloroplast membranes were isolated and stored at 0 to 4/sup 0/C and aliquots were subsequently tested for their retention of energizing capacity. The main factors which increase the stability of this activity were found to be (a) isolation in a potassium-containing medium but storage in a sucrose medium containing a low concentration of electrolytes; (b) the presence of butylated hydroxytoluene (an antioxidant), and a protein such as bovine serum albumin to remove free fatty acids in the medium during storage. Under these conditions, the energization capacity of chloroplasts is retained for more than 40 days.

  20. Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts.

    Science.gov (United States)

    Vasconcelos, A C; Mendiola-Morgenthaler, L R; Floyd, G L; Salisbury, J L

    1976-07-01

    Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.The envelope membranes were separated into two fractions in the gradients according to their banding densities. Electron micrographs showed that the light envelope fraction consisted mostly of single-membrane vesicles, whereas the heavy envelope fraction consisted of multiple layers of folded membranes. Both envelope fractions were ultrastructurally distinct from the thylakoid membranes. PMID:16659627

  1. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    Science.gov (United States)

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  2. Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis)

    Science.gov (United States)

    Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and ...

  3. Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents

    Science.gov (United States)

    Huppe, H. C.; Buchanan, B. B.

    1989-01-01

    A chloroplast type of fructose-1,6-bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiologic value of 8.0 as the Mg2+ concentration was increased from 1 to 16 mM. At 1 mM Mg2+, a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the properties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.

  4. Nucleotide sequence of a spinach chloroplast valine tRNA.

    OpenAIRE

    Sprouse, H M; Kashdan, M; Otis, L; Dudock, B

    1981-01-01

    The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast ...

  5. The action spectrum in chloroplast translocation in multilayer leaf cells

    Directory of Open Access Journals (Sweden)

    Zbigniew Lechowski

    2015-05-01

    Full Text Available By measurement of light transmittance through a leaf as criterion of chloroplast translocation, the action spectrum of Ajuga reptans was established. In the spectrum obtained, a correction was introduced for leaf autoabsorption calculated on the basis of the Beer-Lambert law. The action spectrum has two maxima: at λ= 375 nm and λ= 481 nm. The range above 502 nm has no significant effect on chloroplast translocation. Comparison with other objects examined demonstrated that in multilayer leaf cells riboflavin seems also to be a photoreceptor active in this process.

  6. Biotin Carboxyl Carrier Protein in Barley Chloroplast Membranes

    DEFF Research Database (Denmark)

    Kannangara, C. G.; Jense, C J

    1975-01-01

    Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained by solubil......Biotin localized in barley chloroplast lamellae is covalently bound to a single protein with an approximate molecular weight of 21000. It contains one mole of biotin per mole of protein and functions as a carboxyl carrier in the acetyl-CoA carboxylase reaction. The protein was obtained...

  7. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    Science.gov (United States)

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  8. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  9. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, Agnethe; Jenkins, Tom;

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  10. Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

    Directory of Open Access Journals (Sweden)

    Lung Shiu-Cheung

    2012-03-01

    Full Text Available Abstract Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.

  11. Chloroplast degeneration and its inhibition by kinetin in detached leaves of Cichorium intybus L.

    Directory of Open Access Journals (Sweden)

    F. Młodzianowski

    2015-05-01

    Full Text Available In the chicory (Cichorium intybus L. var. sativum cv. Polanowicka leaves two types of chloroplasts are present differing by their degree of osmiophility of the thylakoid inside. This type of differentiation of chloroplasts has so far been found only in several plant species. The process of chloroplast degeneration in darkness is described. In osmiophilic chloroplasts at certain stage of degeneration minutely layered giant grana were found. Kinetin markedly inhibited the process of chloroplast degeneration, and after prolonged treatment even stimulated the stacking. process of grana thylakoids.

  12. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

    DEFF Research Database (Denmark)

    Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos;

    2016-01-01

    on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals, as well as complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression...

  13. Role of the chloroplast in the predatory dinoflagellate Karlodinium armiger

    DEFF Research Database (Denmark)

    Berge, Terje; Hansen, Per Juel

    2016-01-01

    ABSTRACT: Karlodinium armiger is a phagotrophic dinoflagellate that synthesizes several small chloroplasts of haptophyte origin. It depends on light, but it grows very poorly in standard nutrient growth media (f/2) without food. When fed prey in the light, growth rates increase dramatically (µ=0...

  14. Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

    Science.gov (United States)

    Solti, Adám; Müller, Brigitta; Czech, Viktória; Sárvári, Éva; Fodor, Ferenc

    2014-05-01

    Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

  15. Complete Chloroplast Genome Sequence of Dendrobium nobile from Northeastern India

    Science.gov (United States)

    Parameswaran, Sriram; Sundar, Durai

    2016-01-01

    The orchid species Dendrobium nobile belonging to the family Orchidaceae and genus Dendrobium (a vast genus that encompasses nearly 1,200 species) has an herbal medicinal history of about 2000 years in east and south Asian countries. Here, we report the complete chloroplast genome sequence of D. nobile from northeastern India for the first time.

  16. The complete chloroplast genome sequence of medicinal plant Pinellia ternata.

    Science.gov (United States)

    Han, Limin; Chen, Chen; Wang, Bin; Wang, Zhe-Zhi

    2016-07-01

    Pinellia ternata is an important medicinal plant used in the treatment of cough, to dispel phlegm, to calm vomiting and to terminate early pregnancy, as an anti-ulcer and anti-tumor medicine. In this study, we found that the complete chloroplast genome of Pinellia ternata was 164 013 bp in length, containing a pair of inverted repeats of 25 625 bp separated by a large single-copy region and a small single-copy region of 89 783 bp and 22 980 bp, respectively. The chloroplast genome encodes 132 predicted functional genes, including 87 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The chloroplast DNA is GC-rich (36.7%). The phylogenetic analysis showed a strong sister relationship with Colocasia esculenta, which also strongly supports the position of Pinellia ternata. The complete chloroplast genome sequence of Pinellia ternata reported here has the potential to advance population and phylogenetic studies of this medicinal plant. PMID:26153849

  17. Mitochondrial and chloroplast DNA based phylogeny of Pelargonium (Geraniaceae)

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Pankhurst, C.E.; Gibby, M.

    2000-01-01

    Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as ou

  18. Global Chloroplast Phylogeny and Biogeography of Bracken (Pteridium: Dennstaedtiaceae)

    OpenAIRE

    J.P.;; Thomson, J. A.; Stratford, J. K.; Paul G Wolf

    2009-01-01

    Bracken ferns (genus Pteridium) represent an ancient species complex with a natural worldwide distribution. Pteridium has historically been treated as comprising a single species, but recent treatments have recognized several related species. Phenotypic plasticity, geographically structured morphological variation, and geographically biased sampling have all contributed to taxonomic confusion in the genus. We sampled bracken specimens worldwide and used variable regions of the chloroplast gen...

  19. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae

    OpenAIRE

    Ferreira-Camargo, Livia S; Tran, Miller; Beld, Joris; Burkart, Michael D.; Mayfield, Stephen P

    2015-01-01

    Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly,...

  20. Structure of "Arabidopsis" chloroplastic monothiol glutaredoxin AtGRXcp

    Science.gov (United States)

    Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are conserved across species. "Arabidopsis thaliana" monothiol glutaredoxin AtGRXcp, is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been de...

  1. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    Directory of Open Access Journals (Sweden)

    Cristian A. Carrión

    2014-11-01

    Full Text Available Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs, characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves.

  2. Photosynthetic activity of spinach chloroplasts after isopycnic centrifugation in gradients of silica.

    Science.gov (United States)

    Morgenthaler, J J; Price, C A

    1974-10-01

    Chloroplast suspensions from spinach (Spinacia oleracea L.) were clearly resolved into intact and stripped chloroplasts by isopycnic centrifugation in density gradients of silica sol ("Ludox") and polyethlene glycol. The intact chloroplasts fixed CO(2) and evolved O(2) more rapidly than the crude suspensions; the stripped chloroplasts were inactive. During the photosynthetic fixation of (14)CO(2) in the intact chloroplasts recovered from the gradient, the (14)C label was observed to spread through the photosynthetic intermediate pools, as well as into starch, which indicates that the purified chloroplasts are metabolically competent. This appears to be the first report of the retention of photosynthetic activity following the purification of chloroplasts in density gradients. PMID:16658922

  3. Self-Assembled Enzyme Nanoparticles for Carbon Dioxide Capture.

    Science.gov (United States)

    Shanbhag, Bhuvana Kamath; Liu, Boyin; Fu, Jing; Haritos, Victoria S; He, Lizhong

    2016-05-11

    Enzyme-based processes have shown promise as a sustainable alternative to amine-based processes for carbon dioxide capture. In this work, we have engineered carbonic anhydrase nanoparticles that retain 98% of hydratase activity in comparison to their free counterparts. Carbonic anhydrase was fused with a self-assembling peptide that facilitates the noncovalent assembly of the particle and together were recombinantly expressed from a single gene construct in Escherichia coli. The purified enzymes, when subjected to a reduced pH, form 50-200 nm nanoparticles. The CO2 capture capability of enzyme nanoparticles was demonstrated at ambient (22 ± 2 °C) and higher (50 °C) temperatures, under which the nanoparticles maintain their assembled state. The carrier-free enzymatic nanoparticles demonstrated here offer a new approach to stabilize and reuse enzymes in a simple and cost-effective manner.

  4. Chloroplast Genome Sequence of the Moss Tortula ruralis: Gene Content and Structural Arrangement Relative to Other Green Plant Chloroplast Genomes

    Science.gov (United States)

    Tortula ruralis, a widely distributed moss species in the family Pottiaceae, is increasingly being used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of Tortula ruralis, only the second publishe...

  5. Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.

    Science.gov (United States)

    Tejos, Ricardo I; Mercado, Ana V; Meisel, Lee A

    2010-01-01

    The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

  6. Pb-induced avoidance-like chloroplast movements in fronds of Lemna trisulca L.

    Directory of Open Access Journals (Sweden)

    Sławomir Samardakiewicz

    Full Text Available Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed. An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb2+, 24 h in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H2O2. In addition, we detected an enhanced accumulation of endogenous H2O2 after treatment of plants with lead. Interestingly, H2O2-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H2O2 can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the

  7. Synthesis of 4-(2-substituted hydrazinyl)benzenesulfonamides and their carbonic anhydrase inhibitory effects.

    Science.gov (United States)

    Gul, Halise Inci; Kucukoglu, Kaan; Yamali, Cem; Bilginer, Sinan; Yuca, Hafize; Ozturk, Iknur; Taslimi, Parham; Gulcin, Ilhami; Supuran, Claudiu T

    2016-08-01

    In this study, 4-(2-substituted hydrazinyl)benzenesulfonamides were synthesized by microwave irradiation and their chemical structures were confirmed by (1)H NMR, (13)CNMR, and HRMS. Ketones used were: Acetophenone (S1), 4-methylacetophenone (S2), 4-chloroacetophenone (S3), 4-fluoroacetophenone (S4), 4-bromoacetophenone (S5), 4-methoxyacetophenone (S6), 4-nitroacetophenone (S7), 2-acetylthiophene (S8), 2-acetylfuran (S9), 1-indanone (S10), 2-indanone (S11). The compounds S9, S10 and S11 were reported for the first time, while S1-S8 was synthesized by different method than literature reported using microwave irradiation method instead of conventional heating in this study. The inhibitory effects of 4-(2-substituted hydrazinyl)benzenesulfonamide derivatives (S1-S11) against hCA I and II were studied. Cytosolic hCA I and II isoenzymes were potently inhibited by new synthesized sulphonamide derivatives with Kis in the range of 1.79 ± 0.22-2.73 ± 0.08 nM against hCA I and in the range of 1.72 ± 0.58-11.64 ± 5.21 nM against hCA II, respectively. PMID:26044365

  8. Synthesis of 4-(2-substituted hydrazinyl)benzenesulfonamides and their carbonic anhydrase inhibitory effects.

    Science.gov (United States)

    Gul, Halise Inci; Kucukoglu, Kaan; Yamali, Cem; Bilginer, Sinan; Yuca, Hafize; Ozturk, Iknur; Taslimi, Parham; Gulcin, Ilhami; Supuran, Claudiu T

    2016-08-01

    In this study, 4-(2-substituted hydrazinyl)benzenesulfonamides were synthesized by microwave irradiation and their chemical structures were confirmed by (1)H NMR, (13)CNMR, and HRMS. Ketones used were: Acetophenone (S1), 4-methylacetophenone (S2), 4-chloroacetophenone (S3), 4-fluoroacetophenone (S4), 4-bromoacetophenone (S5), 4-methoxyacetophenone (S6), 4-nitroacetophenone (S7), 2-acetylthiophene (S8), 2-acetylfuran (S9), 1-indanone (S10), 2-indanone (S11). The compounds S9, S10 and S11 were reported for the first time, while S1-S8 was synthesized by different method than literature reported using microwave irradiation method instead of conventional heating in this study. The inhibitory effects of 4-(2-substituted hydrazinyl)benzenesulfonamide derivatives (S1-S11) against hCA I and II were studied. Cytosolic hCA I and II isoenzymes were potently inhibited by new synthesized sulphonamide derivatives with Kis in the range of 1.79 ± 0.22-2.73 ± 0.08 nM against hCA I and in the range of 1.72 ± 0.58-11.64 ± 5.21 nM against hCA II, respectively.

  9. Carbonic Anhydrase IX Expression Correlates with FDG Uptake by Primary Non-Small Cell Lung Cancer

    NARCIS (Netherlands)

    Mees, Gilles; Vangestel, Christel; Dierckx, Rudi; Pauwels, Patrick; Van Meerbeeck, Jan; Van de Wiele, Christophe

    2010-01-01

    Tumor cells are characterized by an increased rate of glucose consumption and glycolysis. This increased glucose consumption leads to tumor acidification, which represents a major obstacle for several therapeutic strategies. Tumor cells have adapted to this acidification by upregulation of several H

  10. Effect of electrostatic interactions on the formation of proton transfer pathways in human carbonic anhydrase II

    Indian Academy of Sciences (India)

    Arijit Roy; Srabani Taraphder

    2007-09-01

    We report here a theoretical study on the effect of electrostatic interactions on the formation of dynamical, proton-conducting hydrogen-bonded networks in the protein HCA II. The conformational fluctuations of His-64 is found to contribute crucially to the mechanism of such path formation irrespective of the way electrostatic interactions are modelled.

  11. Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.

    Science.gov (United States)

    Chan, Kai Xun; Phua, Su Yin; Crisp, Peter; McQuinn, Ryan; Pogson, Barry J

    2016-04-29

    The chloroplast can act as an environmental sensor, communicating with the cell during biogenesis and operation to change the expression of thousands of proteins. This process, termed retrograde signaling, regulates expression in response to developmental cues and stresses that affect photosynthesis and yield. Recent advances have identified many signals and pathways-including carotenoid derivatives, isoprenes, phosphoadenosines, tetrapyrroles, and heme, together with reactive oxygen species and proteins-that build a communication network to regulate gene expression, RNA turnover, and splicing. However, retrograde signaling pathways have been viewed largely as a means of bilateral communication between organelles and nuclei, ignoring their potential to interact with hormone signaling and the cell as a whole to regulate plant form and function. Here, we discuss new findings on the processes by which organelle communication is initiated, transmitted, and perceived, not only to regulate chloroplastic processes but also to intersect with cellular signaling and alter physiological responses. PMID:26735063

  12. The complete chloroplast genome of North American ginseng, Panax quinquefolius.

    Science.gov (United States)

    Han, Zeng-Jie; Li, Wei; Liu, Yuan; Gao, Li-Zhi

    2016-09-01

    We report complete nucleotide sequence of the Panax quinquefolius chloroplast genome using next-generation sequencing technology. The genome size is 156 359 bp, including two inverted repeats (IRs) of 52 153 bp, separated by the large single-copy (LSC 86 184 bp) and small single-copy (SSC 18 081 bp) regions. This cp genome encodes 114 unigenes (80 protein-coding genes, four rRNA genes, and 30 tRNA genes), in which 18 are duplicated in the IR regions. Overall GC content of the genome is 38.08%. A phylogenomic analysis of the 10 complete chloroplast genomes from Araliaceae using Daucus carota from Apiaceae as outgroup showed that P. quinquefolius is closely related to the other two members of the genus Panax, P. ginseng and P. notoginseng. PMID:27158867

  13. The complete chloroplast genome of Torreya fargesii (Taxaceae).

    Science.gov (United States)

    Tao, Ke; Gao, Lei; Li, Jia; Chen, Shanshan; Su, Yingjuan; Wang, Ting

    2016-09-01

    The complete chloroplast genome sequence of Torreya fargesii (Taxaceae), a relic plant endemic to China, is presented in this study. The genome is 137 075 bp in length, with 35.47% average GC content. One copy of the large inverted repeats is lost from this genome. The T. fargesii chloroplast genome encodes 118 unique genes, in which trnI-CAU, trnQ-UUG, trnN-GUU are duplicated. Protein-coding, tRNA and rRNA genes represent 54.7%, 1.9% and 3.4% of the genome, respectively. There are 17 intron-containing genes, of which 6 are tRNA genes. A maximum likelihood phylogenetic analysis revealed a strong sister relationship between Torreya and Amentotaxus. PMID:27158868

  14. The complete chloroplast genome sequence of Fagopyrum cymosum.

    Science.gov (United States)

    Yang, Jun; Lu, Chaolong; Shen, Qi; Yan, Yuying; Xu, Changjiang; Song, Chi

    2016-07-01

    Fagopyrum cymosum is a traditional medicinal plant. In this study, the complete chloroplast genome of Fagopyrum cymosum is presented. The total genome size is 160,546 bp in length, containing a pair of inverted repeats (IRs) of 32,598 bp, separated by large single copy (LSC) and small single copy (SSC) of 84,237 bp and 11,014 bp, respectively. Overall GC contents of the genome were 36.9%. The chloroplast genome harbors 126 annotated genes, including 91 protein coding genes, 29 tRNA genes, and six rRNA genes. Eighteen genes contain one or two introns. Phylogenetic analyses indicated a clear evolutionary relationship among species of Caryophyllales. PMID:26119127

  15. Signal integration by chloroplast phosphorylation networks: An update

    Directory of Open Access Journals (Sweden)

    Anna eSchoenberg

    2012-11-01

    Full Text Available Forty years after the initial discovery of light-dependent protein phosphorylation at the thylakoid membrane system, we are now beginning to understand the roles of chloroplast phosphorylation networks in their function to decode and mediate information on the metabolic status of the organelle to long-term adaptations in plastid and nuclear gene expression. With the help of genetics and functional genomics tools, chloroplast kinases and several hundred phosphoproteins were identified that now await detailed functional characterization. The regulation and the target protein spectrum of some kinases are understood, but this information is fragmentary with respect to kinase and target protein crosstalk in a changing environment. In this review we will highlight the most recent advances in the field and discuss approaches that might lead to a comprehensive understanding of plastid signal integration by protein phosphorylation.

  16. Localized hypermutation and associated gene losses in legume chloroplast genomes

    OpenAIRE

    KAVANAGH, THOMAS; WOLFE, KENNETH; POWELL, ANTOINETTE

    2010-01-01

    PUBLISHED Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual p...

  17. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  18. Study on Chloroplast Ultrastructure in Different Color Period of Euphorbia pulcherrima

    Institute of Scientific and Technical Information of China (English)

    FU Jia; NIU De; WANG Lijuan

    2008-01-01

    By the observation of chloroplast ultrastructure in different period of bract colors of Euphorbia pulcherrima,the paper studied the change of chloroplast ultrastructrural in the transition process of bract colors, identified the rehtionship between E.pulcherrima color change and the chloroplast ultrastructure to provide theorical bases for the cultivation management and further study of E.pulcherrima.Ultrastructural study showed that in the process of change from green to red,the chloroplast of bracts disintegrated gradually,lamellar structure was destroyed gradually,and the content of chloroplasts in mesophyll cells was also reduced gradually. When bracts color resumed to turn green gradually,the content of chloroplasts in mesophyll cells was also increased gradually.

  19. Carbon isotopes in mollusk shell carbonates

    Science.gov (United States)

    McConnaughey, Ted A.; Gillikin, David Paul

    2008-10-01

    Mollusk shells contain many isotopic clues about calcification physiology and environmental conditions at the time of shell formation. In this review, we use both published and unpublished data to discuss carbon isotopes in both bivalve and gastropod shell carbonates. Land snails construct their shells mainly from respired CO2, and shell δ13C reflects the local mix of C3 and C4 plants consumed. Shell δ13C is typically >10‰ heavier than diet, probably because respiratory gas exchange discards CO2, and retains the isotopically heavier HCO3 -. Respired CO2 contributes less to the shells of aquatic mollusks, because CO2/O2 ratios are usually higher in water than in air, leading to more replacement of respired CO2 by environmental CO2. Fluid exchange with the environment also brings additional dissolved inorganic carbon (DIC) into the calcification site. Shell δ13C is typically a few ‰ lower than ambient DIC, and often decreases with age. Shell δ13C retains clues about processes such as ecosystem metabolism and estuarine mixing. Ca2+ ATPase-based models of calcification physiology developed for corals and algae likely apply to mollusks, too, but lower pH and carbonic anhydrase at the calcification site probably suppress kinetic isotope effects. Carbon isotopes in biogenic carbonates are clearly complex, but cautious interpretation can provide a wealth of information, especially after vital effects are better understood.

  20. Chloroplast quality control - balancing energy production and stress.

    Science.gov (United States)

    Woodson, Jesse D

    2016-10-01

    Contents 36 I. 36 II. 37 III. 37 IV. 38 V. 39 VI. 40 VII. 40 40 References 40 SUMMARY: All organisms require the ability to sense their surroundings and adapt. Such capabilities allow them to thrive in a wide range of habitats. This is especially true for plants, which are sessile and have to be genetically equipped to withstand every change in their environment. Plants and other eukaryotes use their energy-producing organelles (i.e. mitochondria and chloroplasts) as such sensors. In response to a changing cellular or external environment, these organelles can emit 'retrograde' signals that alter gene expression and/or cell physiology. This signaling is important in plants, fungi, and animals and impacts diverse cellular functions including photosynthesis, energy production/storage, stress responses, growth, cell death, ageing, and tumor progression. Originally, chloroplast retrograde signals in plants were known to lead to the reprogramming of nuclear transcription. New research, however, has pointed to additional posttranslational mechanisms that lead to chloroplast regulation and turnover in response to stress. Such mechanisms involve singlet oxygen, ubiquitination, the 26S proteasome, and cellular degradation machinery. PMID:27533783

  1. Chloroplast ultrastructure in leaves of Cucumis sativus chlorophyll mutant

    Directory of Open Access Journals (Sweden)

    Irena Palczewska

    2014-02-01

    Full Text Available The developing and young leaves of Cucumis sativus chlorophyll mutants are yellow, when mature they become green and do not differ in their colour from those of control plants. The mesophyll of yellow leaves contains a diversiform plastid population with a varying degree of defectiveness, which is mainly manifested in the reduction or disorganization of the typical thylakoid system. DNA areas, ribosome-like particles and aggregates of electron-dense material are preserved in the stroma of mutated plastids. Starch grains are deficient. Apart from mutated plastids, chloroplasts with a normal structure, as in control plants, were also observed.The leaf greening process is accompanied by a reconstruction and rearrangement of the inner chloroplast lamellar system and an ability to accumulate starch. However, in the mutant chloroplasts as compared with control-plant ones, an irregular arrangement of grana and reduced number of inter-grana thylakoids can be seen. An osmiophilic substance stored in the stroma of mutated plastids and the vesicles formed from an internal plastid membrane take part in restoration of the membrane system.

  2. Chloroplast retrograde regulation of heat stress responses in plants

    Directory of Open Access Journals (Sweden)

    Ai-Zhen eSun

    2016-03-01

    Full Text Available It is well known that intracellular signaling from chloroplast to nucleus plays a vital role in stress responses to survive environmental perturbations. The chloroplasts were proposed as sensors to heat stress since components of the photosynthetic apparatus housed in the chloroplast are the primary susceptible targets of thermal damage in plants. Thus, communicating subcellular perturbations to the nucleus is critical during exposure to extreme environmental conditions such as heat stress. By coordinating expression of stress specific nuclear genes essential for adaptive responses to hostile environment, plants optimize different cell functions and activate acclimation responses through retrograde signaling pathways. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. In recent years several putative retrograde signals released from plastids that regulate nuclear genes have been identified and signaling pathways have been proposed. In this review, we provide an update on retrograde signals derived from tetrapyrroles, carotenoids, reactive oxygen species (ROS and organellar gene expression (OGE in the context of heat stress responses and address their roles in retrograde regulation of heat-responsive gene expression, systemic acquired acclimation and cellular coordination in plants.

  3. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    Science.gov (United States)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  4. Chloroplast Retrograde Regulation of Heat Stress Responses in Plants.

    Science.gov (United States)

    Sun, Ai-Zhen; Guo, Fang-Qing

    2016-01-01

    It is well known that intracellular signaling from chloroplast to nucleus plays a vital role in stress responses to survive environmental perturbations. The chloroplasts were proposed as sensors to heat stress since components of the photosynthetic apparatus housed in the chloroplast are the major targets of thermal damage in plants. Thus, communicating subcellular perturbations to the nucleus is critical during exposure to extreme environmental conditions such as heat stress. By coordinating expression of stress specific nuclear genes essential for adaptive responses to hostile environment, plants optimize different cell functions and activate acclimation responses through retrograde signaling pathways. The efficient communication between plastids and the nucleus is highly required for such diverse metabolic and biosynthetic functions during adaptation processes to environmental stresses. In recent years, several putative retrograde signals released from plastids that regulate nuclear genes have been identified and signaling pathways have been proposed. In this review, we provide an update on retrograde signals derived from tetrapyrroles, carotenoids, reactive oxygen species (ROS) and organellar gene expression (OGE) in the context of heat stress responses and address their roles in retrograde regulation of heat-responsive gene expression, systemic acquired acclimation, and cellular coordination in plants. PMID:27066042

  5. CDP1, a novel component of chloroplast division site positioning system in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Min Zhang; Yong Hu; Jingjing Jia; Dapeng Li; Runjie Zhang; Hongbo Gao; Yikun He

    2009-01-01

    Chloroplasts are plant-specific organelles that evolved from endosymbiotic cyanobacteria. They divide through binary fission. Selection of the chloroplast division site is pivotal for the symmetric chloroplast division. In E. coli, positioning of the division site at the midpoint of the cell is regulated by dynamic oscillation of the Min system, which includes MinC, MinD and MinE. Homologs of Mind and MinE in plants are involved in chloroplast division. The homolog of MinC still has not been identified in higher plants. However, an FtsZ-like protein, ARC3, was found to be involved in chloroplast division site positioning. Here, we report that chloroplast division site positioning 1 (AtCDP1) is a novel chloroplast division protein involved in chloroplast division site placement in Arabidopsis. AtCDP1 was dis-covered by screening an Arabidopsis cDNA expression library in bacteria for colonies with a cell division phenotype. AtCDP1 is exclusively expressed in young green tissues in Arabidopsis. Elongated chloroplasts with multiple division sites were observed in the loss-of-function cdpl mutant. Overexpression of AtCDPI caused a chloroplast division phe-notype too. Protein interaction assays suggested that AtCDP1 may mediate the chloroplast division site positioning through the interaction with ARC3. Overall, our results indicate that AtCDP1 is a novel component of the chloroplast division site positioning system, and the working mechanism of this system is different from that of the traditional MinCDE system in prokaryotic cells.

  6. Combined effects of light and water stress on chloroplast volume regulation.

    OpenAIRE

    McCain, D.C.

    1995-01-01

    A nuclear magnetic resonance technique was used to measure changes in the water content of Acer platanoides chloroplasts in leaf discs that had reached osmotic equilibrium with external solutions either in the dark or under exposure to light. Results showed that chloroplast volume regulation (CVR) maintained constant water content in the chloroplasts over a range of water potentials in the dark, but CVR failed when the water potential fell below a critical value. The critical potential was lo...

  7. Longevity of guard cell chloroplasts in falling leaves: implication for stomatal function and cellular aging

    Energy Technology Data Exchange (ETDEWEB)

    Zeiger, E.; Schwartz, A.

    1982-11-12

    Guard cell chloroplasts in senescing leaves from 12 species of perennial trees and three species of annual plants survived considerably longer than their mesophyll counterparts. In Ginkgo biloba, stomata from yellow leaves opened during the day and closed at night; guard cell chloroplasts from these leaves showed fluorescence transients associated with electron transport and photophosphorylation. These findings indicate that guard cell chloroplasts are highly conserved throughout the life-span of the leaf and that leaves retain stomatal control during senescence.

  8. Glucose-1-phosphate transport into protoplasts and chloroplasts from leaves of Arabidopsis.

    Science.gov (United States)

    Fettke, Joerg; Malinova, Irina; Albrecht, Tanja; Hejazi, Mahdi; Steup, Martin

    2011-04-01

    Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.

  9. Chloroplast transformation for engineering of photosynthesis.

    Science.gov (United States)

    Hanson, Maureen R; Gray, Benjamin N; Ahner, Beth A

    2013-01-01

    Many efforts are underway to engineer improvements in photosynthesis to meet the challenges of increasing demands for food and fuel in rapidly changing environmental conditions. Various transgenes have been introduced into either the nuclear or plastid genomes in attempts to increase photosynthetic efficiency. We examine the current knowledge of the critical features that affect levels of expression of plastid transgenes and protein accumulation in transplastomic plants, such as promoters, 5' and 3' untranslated regions, RNA-processing sites, translation signals and amino acid sequences that affect protein turnover. We review the prior attempts to manipulate the properties of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through plastid transformation. We illustrate how plastid operons could be created for expression of the multiple genes needed to introduce new pathways or enzymes to enhance photosynthetic rates or reduce photorespiration. We describe here the past accomplishments and future prospects for manipulating plant enzymes and pathways to enhance carbon assimilation through plastid transformation. PMID:23162121

  10. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Angelova, Angelina [University of Arizona; Park, Sang-Hycuk [University of Arizona; Kyndt, John [Bellevue University; Fitzsimmons, Kevin [University of Arizona; Brown, Judith K [University of Arizona

    2013-09-01

    With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

  11. Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1997-06-17

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  12. Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum.

    Science.gov (United States)

    Walbot, V

    1977-07-01

    The first method for the direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C(4) plant is described. Centrifugation of mixed chloroplast preparations from Panicum maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are judged to be bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels, and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceralde-hyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to deoxyribonuclease. Chloroplast enzyme activities are inhibited by silica sol due to the Mg(2+) chelating activity of this reagent. However, well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used. PMID:16660019

  13. ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

    Science.gov (United States)

    Nomura, Yuhta; Takabayashi, Taito; Kuroda, Hiroshi; Yukawa, Yasushi; Sattasuk, Kwanchanok; Akita, Mitsuru; Nozawa, Akira; Tozawa, Yuzuru

    2012-01-01

    Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

  14. Biomimetic sequestration of CO2 in carbonate form: Role of produced waters and other brines

    International Nuclear Information System (INIS)

    The objective of our research is the development of a system resembling a CO2 scrubber, in which carbonic anhydrase catalyzes the rate of CO2 hydration for subsequent fixation into stable mineral carbonates. The present focus is on the use of produced waters as one of various possible sources of the counterions for precipitation. Carbonate precipitation has been studied from synthetic brines corresponding to a range of compositions of produced waters from the Permian and San Juan Basins, and promising results have been obtained with carbonic anhydrase. CO2 sequestration capacities are estimated to be 0.49-1.85x103 tonnes CO2/year and 1.28-2.80x105 tonnes CO2/year for the San Juan and Permian Basins, respectively, per cycle, up to ∼3mt CO2 per year total, based on the volumes of produced waters in 2002. (author)

  15. Covalent enzyme immobilization onto carbon nanotubes using a membrane reactor

    Science.gov (United States)

    Voicu, Stefan Ioan; Nechifor, Aurelia Cristina; Gales, Ovidiu; Nechifor, Gheorghe

    2011-05-01

    Composite porous polysulfone-carbon nanotubes membranes were prepared by dispersing carbon nanotubes into a polysulfone solution followed by the membrane formation by phase inversion-immersion precipitation technique. The carbon nanotubes with amino groups on surface were functionalized with different enzymes (carbonic anhydrase, invertase, diastase) using cyanuric chloride as linker between enzyme and carbon nanotube. The composite membrane was used as a membrane reactor for a better dispersion of carbon nanotubes and access to reaction centers. The membrane also facilitates the transport of enzymes to active carbon nanotubes centers for functionalization (amino groups). The functionalized carbon nanotubes are isolated by dissolving the membranes after the end of reaction. Carbon nanotubes with covalent immobilized enzymes are used for biosensors fabrications. The obtained membranes were characterized by Scanning Electron Microscopy, Thermal analysis, FT-IR Spectroscopy, Nuclear Magnetic Resonance, and functionalized carbon nanotubes were characterized by FT-IR spectroscopy.

  16. Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

    Directory of Open Access Journals (Sweden)

    Yoko Kimata-Ariga

    Full Text Available Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT, nitrite reductase (NiR and glutamine synthetase (GS, separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE. GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa and multiple sizes (>120 kDa, respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

  17. Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis

    OpenAIRE

    Kodama, Yutaka; Suetsugu, Noriyuki; Kong, Sam-Geun; Wada, Masamitsu

    2010-01-01

    Chloroplasts move toward weak light (accumulation response) and away from strong light (avoidance response). The fast and accurate movement of chloroplasts in response to ambient light conditions is essential for efficient photosynthesis and photodamage prevention in chloroplasts. Here, we report that two Arabidopsis mutants, weak chloroplast movement under blue light 1 (web1) and web2, are defective in both the avoidance and the accumulation responses. Map-based cloning revealed that both ge...

  18. A comparative approach to elucidate chloroplast genome replication

    Directory of Open Access Journals (Sweden)

    Krishnan Neeraja M

    2009-05-01

    Full Text Available Abstract Background Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts. Results We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC and the Large Single Copy (LSC regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication. Conclusion Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population

  19. Analysis of Acorus calamus chloroplast genome and its phylogenetic implications.

    Science.gov (United States)

    Goremykin, Vadim V; Holland, Barbara; Hirsch-Ernst, Karen I; Hellwig, Frank H

    2005-09-01

    Determining the phylogenetic relationships among the major lines of angiosperms is a long-standing problem, yet the uncertainty as to the phylogenetic affinity of these lines persists. While a number of studies have suggested that the ANITA (Amborella-Nymphaeales-Illiciales-Trimeniales-Aristolochiales) grade is basal within angiosperms, studies of complete chloroplast genome sequences also suggested an alternative tree, wherein the line leading to the grasses branches first among the angiosperms. To improve taxon sampling in the existing chloroplast genome data, we sequenced the chloroplast genome of the monocot Acorus calamus. We generated a concatenated alignment (89,436 positions for 15 taxa), encompassing almost all sequences usable for phylogeny reconstruction within spermatophytes. The data still contain support for both the ANITA-basal and grasses-basal hypotheses. Using simulations we can show that were the ANITA-basal hypothesis true, parsimony (and distance-based methods with many models) would be expected to fail to recover it. The self-evident explanation for this failure appears to be a long-branch attraction (LBA) between the clade of grasses and the out-group. However, this LBA cannot explain the discrepancies observed between tree topology recovered using the maximum likelihood (ML) method and the topologies recovered using the parsimony and distance-based methods when grasses are deleted. Furthermore, the fact that neither maximum parsimony nor distance methods consistently recover the ML tree, when according to the simulations they would be expected to, when the out-group (Pinus) is deleted, suggests that either the generating tree is not correct or the best symmetric model is misspecified (or both). We demonstrate that the tree recovered under ML is extremely sensitive to model specification and that the best symmetric model is misspecified. Hence, we remain agnostic regarding phylogenetic relationships among basal angiosperm lineages.

  20. Functional proteomics of barley and barley chloroplasts – strategies, methods and perspectives

    DEFF Research Database (Denmark)

    Petersen, Jørgen; Rogowska-Wrzesinska, Adelina; Jensen, Ole Nørregaard

    2013-01-01

    tolerance, micronutrient utilization, and photosynthesis in barley. In the present review we present the current state of proteomics research for investigations of barley chloroplasts, i.e., the organelle that contain the photosynthetic apparatus in the plant. We describe several different proteomics...... strategies and discuss their applications in characterization of the barley chloroplast as well as future perspectives for functional proteomics in barley research....

  1. The Unicellular Green Alga Chlamydomonas reinhardtii as an Experimental System to Study Chloroplast RNA Metabolism

    Science.gov (United States)

    Nickelsen, J.; Kück, U.

    Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

  2. Treatment with antibiotics that interfere with peptidoglycan biosynthesis inhibits chloroplast division in the desmid Closterium.

    Directory of Open Access Journals (Sweden)

    Hiroko Matsumoto

    Full Text Available Charophytes is a green algal group closely related to land plants. We investigated the effects of antibiotics that interfere with peptidoglycan biosynthesis on chloroplast division in the desmid Closterium peracerosum-strigosum-littorale complex. To detect cells just after division, we used colchicine, which inhibits Closterium cell elongation after division. Although normal Closterium cells had two chloroplasts before and after cell division, cells treated with ampicillin, D-cycloserine, or fosfomycin had only one chloroplast after cell division, suggesting that the cells divided without chloroplast division. The antibiotics bacitracin and vancomycin showed no obvious effect. Electron microscopic observation showed that irregular-shaped chloroplasts existed in ampicillin-treated Closterium cells. Because antibiotic treatments resulted in the appearance of long cells with irregular chloroplasts and cell death, we counted cell types in the culture. The results suggested that cells with one chloroplast appeared first and then a huge chloroplast was generated that inhibited cell division, causing elongation followed by cell death.

  3. Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle

    DEFF Research Database (Denmark)

    Träger, Chantal; Rosenblad, Magnus Alm; Ziehe, Dominik;

    2012-01-01

    The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of ...

  4. Contrasting modes of inorganic carbon acquisition amongst Symbiodinium (Dinophyceae) phylotypes.

    Science.gov (United States)

    Brading, Patrick; Warner, Mark E; Smith, David J; Suggett, David J

    2013-10-01

    Growing concerns over ocean acidification have highlighted the need to critically understand inorganic carbon acquisition and utilization in marine microalgae. Here, we contrast these characteristics for the first time between two genetically distinct dinoflagellate species of the genus Symbiodinium (phylotypes A13 and A20) that live in symbiosis with reef-forming corals. Both phylotypes were grown in continuous cultures under identical environmental conditions. Rubisco was measured using quantitative Western blots, and radioisotopic (14) C uptake was used to characterize light- and total carbon dioxide (TCO2 )-dependent carbon fixation, as well as inorganic carbon species preference and external carbonic anhydrase activity. A13 and A20 exhibited similar rates of carbon fixation despite cellular concentrations of Rubisco being approximately four-fold greater in A13. The uptake of CO2 over HCO3 - was found to support the majority of carbon fixation in both phylotypes. However, A20 was also able to indirectly utilize HCO3 - by first converting it to CO2 via external carbonic anhydrase. These results show that adaptive differences in inorganic carbon acquisition have evolved within the Symbiodinium genus, which thus carries fundamental implications as to how this functionally key genus will respond to ocean acidification, but could also represent a key trait factor that influences their productivity when in hospite of their coral hosts.

  5. The complete chloroplast genome sequence of Curcuma flaviflora (Curcuma).

    Science.gov (United States)

    Zhang, Yan; Deng, Jiabin; Li, Yangyi; Gao, Gang; Ding, Chunbang; Zhang, Li; Zhou, Yonghong; Yang, Ruiwu

    2016-09-01

    The complete chloroplast (cp) genome of Curcuma flaviflora, a medicinal plant in Southeast Asia, was sequenced. The genome size was 160 478 bp in length, with 36.3% GC content. A pair of inverted repeats (IRs) of 26 946 bp were separated by a large single copy (LSC) of 88 008 bp and a small single copy (SSC) of 18 578 bp, respectively. The cp genome contained 132 annotated genes, including 79 protein coding genes, 30 tRNA genes, and four rRNA genes. And 19 of these genes were duplicated in inverted repeat regions. PMID:26367332

  6. Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

    Directory of Open Access Journals (Sweden)

    Randhir K Bharti

    Full Text Available A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO. The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC, however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials.

  7. Chloroplast transformation of Platymonas (Tetraselmis subcordiformis with the bar gene as selectable marker.

    Directory of Open Access Journals (Sweden)

    Yulin Cui

    Full Text Available The objective of this research was to establish a chloroplast transformation technique for Platymonas (Tetraselmis subcordiformis. Employing the gfp gene as a reporter and the bar gene as a selectable marker, transformation vectors of P. subcordiformis chloroplast were constructed with endogenous fragments rrn16S-trnI (left and trnA-rrn23S (right as a recombination site of the chloroplast genome. The plasmids were transferred into P. subcordiformis via particle bombardment. Confocal laser scanning microscopy indicated that the green fluorescence protein was localized in the chloroplast of P. subcordiformis, confirming the activity of the Chlamydomonas reinhardtii promoter. Cells transformed with the bar gene were selected using the herbicide Basta. Resistant colonies were analyzed by PCR and Southern blotting, and the results indicated that the bar gene was successfully integrated into the chloroplast genome via homologous recombination. The technique will improve genetic engineering of this alga.

  8. Update on Chloroplast Research: New Tools, New Topics, and New Trends

    Institute of Scientific and Technical Information of China (English)

    Ute Armbruster; Paolo Pesaresi; Mathias Pribil; Alexander Hertle; Dario Leister

    2011-01-01

    Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional characterization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowledge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire complement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants.

  9. YGL9, encoding the putative chloroplast signal recognition particle 43 kDa protein in rice, is involved in chloroplast development

    Institute of Scientific and Technical Information of China (English)

    WANG Zhong-wei; LI Yun-feng; LING Ying-hua; SANG Xian-chun; HE Guang-hua; ZHANG Tian-quan; XING Ya-di; ZENG Xiao-qin; WANG Ling; LIU Zhong-xian; SHI Jun-qiong; ZHU Xiao-yan; MA Ling

    2016-01-01

    The nuclear-encoded light-harvesting chlorophyla/b-binding proteins (LHCPs) are speciifcaly translocated from the stroma into the thylakoid membrane through the chloroplast signal recognition particle (cpSRP) pathway. The cpSRP is composed of a cpSRP43 protein and a cpSRP54 protein, and it forms a soluble transit complex with LHCP in the chloroplast stroma. Here, we identiifed theYGL9gene that is predicted to encode the probable rice cpSRP43 protein from a rice yelow-green leaf mutant. A phylogenetic tree showed that an important conserved protein family, cpSRP43, is present in almost al green photosynthetic organisms such as higher plants and green algae. Sequence analysis showed that YGL9 comprises a chloroplast transit peptide, three chromodomains and four ankyrin repeats, and the chromodomains and ankyrin repeats are probably involved in protein-protein interactions. Subcelular localization showed that YGL9 is localized in the chloroplast. Expression pattern analysis indicated thatYGL9is mainly expressed in green leaf sheaths and leaves. Quantitative real-time PCR analysis showed that the expression levels of genes associated with pigment metabolism, chloroplast development and photosynthesis were distinctly affected in theygl9mutant. These results indicated thatYGL9 is possibly involved in pigment metabolism, chloroplast development and photosynthesis in rice.

  10. Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

    Directory of Open Access Journals (Sweden)

    Wolf Paul G

    2010-02-01

    Full Text Available Abstract Background Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. Results The Tortula chloroplast genome is ~123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the ~71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. Conclusions Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

  11. The complete chloroplast genome provides insight into the evolution and polymorphism of Panax ginseng

    Directory of Open Access Journals (Sweden)

    Yongbing eZhao

    2015-01-01

    Full Text Available Panax ginseng C.A. Meyer (P. ginseng is an important medicinal plant and is often used in traditional Chinese medicine. With next generation sequencing (NGS technology, we determined the complete chloroplast genome sequences for four Chinese P. ginseng strains, which are Damaya (DMY, Ermaya (EMY, Gaolishen (GLS and Yeshanshen (YSS. The total chloroplast genome sequence length for DMY, EMY and GLS was 156,354 bp, while that for YSS was 156,355 bp. Comparative genomic analysis of the chloroplast genome sequences indicate that gene content, GC content, and gene order in DMY are quite similar to its relative species, and nucleotide sequence diversity of inverted repeat region (IR is lower than that of its counterparts, large single copy region (LSC and small single copy region (SSC. A comparison among these four P. ginseng strains revealed that the chloroplast genome sequences of DMY, EMY, and GLS were identical and YSS had a 1-bp insertion at base 5472. To further study the heterogeneity in chloroplast genome during domestication, high-resolution reads were mapped to the genome sequences to investigate the differences at the minor allele level; 208 minor allele sites with minor allele frequencies (MAF of ≥ 0.05 were identified. The polymorphism site numbers per kb of chloroplast genome sequence for DMY, EMY, GLS, and YSS were 0.74, 0.59, 0.97, and 1.23, respectively. All the minor allele sites located in LSC and IR regions, and the four strains showed the same variation types (substitution base or indel at all identified polymorphism sites. Comparison results of heterogeneity in the chloroplast genome sequences showed that the minor allele sites on the chloroplast genome were undergoing purifying selection to adapt to changing environment during domestication process. A study of P. ginseng chloroplast genome with particular focus on minor allele sites would aid in investigating the dynamics on the chloroplast genomes and different P. ginseng

  12. The complete chloroplast genome of banana (Musa acuminata, Zingiberales: insight into plastid monocotyledon evolution.

    Directory of Open Access Journals (Sweden)

    Guillaume Martin

    Full Text Available BACKGROUND: Banana (genus Musa is a crop of major economic importance worldwide. It is a monocotyledonous member of the Zingiberales, a sister group of the widely studied Poales. Most cultivated bananas are natural Musa inter-(sub-specific triploid hybrids. A Musa acuminata reference nuclear genome sequence was recently produced based on sequencing of genomic DNA enriched in nucleus. METHODOLOGY/PRINCIPAL FINDINGS: The Musa acuminata chloroplast genome was assembled with chloroplast reads extracted from whole-genome-shotgun sequence data. The Musa chloroplast genome is a circular molecule of 169,972 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,338 bp and a Small Single Copy region (SSC, 10,768 bp separated by Inverted Repeat regions (IRs, 35,433 bp. Two forms of the chloroplast genome relative to the orientation of SSC versus LSC were found. The Musa chloroplast genome shows an extreme IR expansion at the IR/SSC boundary relative to the most common structures found in angiosperms. This expansion consists of the integration of three additional complete genes (rps15, ndhH and ycf1 and part of the ndhA gene. No such expansion has been observed in monocots so far. Simple Sequence Repeats were identified in the Musa chloroplast genome and a new set of Musa chloroplastic markers was designed. CONCLUSION: The complete sequence of M. acuminata ssp malaccensis chloroplast we reported here is the first one for the Zingiberales order. As such it provides new insight in the evolution of the chloroplast of monocotyledons. In particular, it reinforces that IR/SSC expansion has occurred independently several times within monocotyledons. The discovery of new polymorphic markers within Musa chloroplast opens new perspectives to better understand the origin of cultivated triploid bananas.

  13. The molecular architecture of the chloroplast thylakoid membrane

    Energy Technology Data Exchange (ETDEWEB)

    Stefansson, H.

    1996-08-01

    Non-detergent procedure for isolation of sub-thylakoid vesicle populations derived from different structural domains of the chloroplast thylakoid membrane has been developed. Sub-thylakoid vesicles representing the grana, grana core, stroma lamellae, and the grana margins have been isolated and their protein composition has been investigated. Furthermore a novel non-detergent procedure for investigating the pigment composition of photosynthetic complexes located in the different structural domains has been developed. This procedure circumvents selective extractions, an perturbing effect often combined with detergent isolations of membrane bound protein complexes. The fractionation experiments show that the NADPH dehydrogenase, suggested to operate as NADPH or ferredoxin-plastoquinone oxidoreductase in cyclic electron transport around photosystem I, is stoichiometrically depleted on photosystem I basis in the grana domain. The fractionation studies are consistent with the model of the thylakoid membrane where the photosystems in the grana are operating in a linear electron transport whereas the site of cyclic electron transport is in the stroma lamellae. It is suggested that partial destacking of grana, as a result of light-induced protein phosphorylation, may promote the exposure of the granal photosystem I centers to the chloroplast stroma and thereby enhance their participation in cyclic electron transport activity. 146 refs, 18 figs

  14. The complete chloroplast genome of Cinnamomum kanehirae Hayata (Lauraceae).

    Science.gov (United States)

    Wu, Chia-Chen; Ho, Cheng-Kuen; Chang, Shu-Hwa

    2016-07-01

    The complete chloroplast genome of Cinnamomum kanehirae (Hayata), the first to be completely sequenced of Lauraceae family, is presented in this study. The total genome size is 152,700 bp, with a typical circular structure including a pair of inverted repeats (IRa/b) of 20,107 bp of length separated by a large single-copy region (LSC) and a small single-copy region (SSC) of 93,642 bp and 18,844 bp of length, respectively. The overall GC content of the genome is 39.1%. The nucleotide sequence shows 91% identities with Liriodendron tulipifera in the Magnoliaceae. In total, 123 annotated genes consisted of 79 coding genes, eight rRNA genes, and 36 tRNA genes. Among all 79 coding genes, seven genes (rpoC1, atpF, rpl2, ndhB, ndhA, rps16, and rpl2) contain one intron, while two genes (ycf3 and clpP) contain two introns. The maximum likelihood phylogenetic analysis revealed that C. kanehirae chloroplast genome is closely related to Calycanthus fertilis within Laurales order. PMID:26053940

  15. Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

    Science.gov (United States)

    Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

    2009-07-01

    Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast

  16. [Effects of exogenous silicon on active oxygen scavenging systems in chloroplasts of cucumber (Cucumis sativus L.) seedlings under salt stress].

    Science.gov (United States)

    Qian, Qiong-Qiu; Zai, Wen-San; Zhu, Zhu-Jun; Yu, Jing-Quan

    2006-02-01

    With K(2)SiO(4) (1.0 mmol/L) treatment, the effects of Si on the distribution of Na(+), K(+) to chloroplasts and antioxidant system of cucumber leaves under 50 mmol/L NaCl stress were studied. The results showed that there was a selective transport of K(+) into the chloroplasts so that Na(+) content of chloroplasts was lower under Si treatment (Table 1); H(2)O(2) and MDA contents in chloroplasts were significantly decreased (Fig.1), and the activities of SOD, APX, GR and DHAR were increased simultaneity (Fig.2), and AsA, GSH contents were also increased in chloroplasts of salt-stressed cucumber by additional Si treatment (Fig.3). It may be concluded that Si could decrease absorption of Na(+) and increase ability of active oxygen scavenging in chloroplasts, therefore the injury of chloroplast membrane under salinity stress in cucumber was alleviated. PMID:16477139

  17. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    Directory of Open Access Journals (Sweden)

    Oldenburg Delene J

    2007-03-01

    Full Text Available Abstract Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI, SYBR Green I (SG, SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.

  18. Is Chloroplast Movement in Tobacco Plants Influenced Systemically after Local Illumination or Burning Stress?

    Institute of Scientific and Technical Information of China (English)

    Jan Naus; Monika Rolencova; Vladimira Hlavackova

    2008-01-01

    Chloroplast movement has been studied In many plants mainly in relation to the local light, mechanical or stress effects. Here we investigated possible systemic responses of chloroplast movement to local light or burning stress in tobacco plants (Nicotiana tabacum cv. Samsun). Chloroplast movement was measured using two independent methods: one with a SPAD 502 Chlorophyll meter and another by collimated transmittance at a selected wavelength (676 nm). A sensitive pedodic movement of chloroplasts was used in high or low (2 000 or 50 μmol/m2 per s photosynthetically active radiation, respectively) cold white light with periods of 50 or 130 min. Measurements were carried out in the irradiated area, in the non-irradiated area of the same leaf or in the leaf located on the stem below the irradiated or burned one. No significant changes in systemic chloroplast movement in non-irradiated parts of the leaf and in the non-treated leaf were detected. Our data indicate that chloroplast movement in tobacco is dependent dominantly on the intensity and spectral composition of the incident light and on the local stimulation and state of the target tissue. No systemic signal was strong enough tovoke a detectable systemic response in chloroplast movement in distant untreated tissues of tobacco plants.

  19. Abscisic acid and blue light signaling pathways in chloroplast movements in Arabidopsis mesophyll.

    Science.gov (United States)

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Banaś, Agnieszka Katarzyna; Janowiak, Franciszek; Gabryś, Halina

    2016-01-01

    Abscisic acid (ABA) and phototropins act antagonistically to control stomatal movements. Here, we investigated the role of ABA in phototropin-directed chloroplast movements in mesophyll cells of Arabidopsis thaliana. We analyzed the expression of phototropins at mRNA and protein level under the influence of ABA. PHOT1 mRNA level was decreased by ABA in the dark while it was insensitive to ABA in light. PHOT2 mRNA level was independent of the hormone treatment. The levels of phototropin proteins were down-regulated by ABA, both in darkness and light. No impact of exogenous ABA on amplitudes and kinetics of chloroplast movements was detected. Chloroplast responses in wild type Arabidopsis and three mutants, abi4, abi2 (abscisic acid insensitive4, 2) and aba1 (abscisic acid1), were measured to account for endogenous ABA signaling. The chloroplast responses were slightly reduced in abi2 and aba1 mutants in strong light. To further investigate the effect, abi2 and aba1 mutants were supplemented with exogenous ABA. In the aba1 mutant, the reaction was rescued but in abi2 it was unaffected. Our results show that ABA is not directly involved in phototropin-controlled chloroplast responses in mature leaves of Arabidopsis. However, the disturbance of ABA biosynthesis and signaling in mutants affects some elements of the chloroplast movement mechanism. In line with its role as a stress hormone, ABA appears to enhance plant sensitivity to light and promote the chloroplast avoidance response.

  20. A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited

    Energy Technology Data Exchange (ETDEWEB)

    Sutton, C.A.; Hanson, M.R. [Cornell Univ., Ithaca, NY (United States); Zoubenko, O.V.; Maliga, P. [State Univ. of New Jersey, Piscataway, NJ (United States)

    1995-03-01

    RNA editing occurs in two higher-plant organelles, chloroplasts, and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components. 33 refs., 5 figs.

  1. The Chloroplast Outer Envelope Membrane: The Edge of Light and Excitement

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The chloroplast is surrounded by a double-membrane envelope at which proteins, ions, and numerous metabolites Including nucleotides, amino acids, fatty acids, and carbohydrates are exchanged between the two aqueous phases, the cytoplasm and the chloroplast stroma. The chloroplast envelope is also the location where the biosynthesis and accumulation of various lipids take place. By contrast to the inner membrane, which contains a number of specific transporters and acts as the permeability barrier, the chloroplast outer membrane has often been considered a passive compartment derived from the phagosomal membrane. However, the presence of galactoglycerolipids and β-barrel membrane proteins support the common origin of the outer membranes of the chloroplast envelope and extant cyanobacteria. Furthermore, recent progress in the field underlines that the chloroplast outer envelope plays important roles not only for translocation of various molecules, but also for regulation of metabolic activities and signaling processes. The chloroplast outer envelope membrane offers various interesting and challenging questions that are relevant to the understanding of organelle biogenesis, plant growth and development, and also membrane biology in general.

  2. The chloroplast genome of a symbiodinium sp. clade C3 isolate

    KAUST Repository

    Barbrook, Adrian C.

    2014-01-01

    Dinoflagellate algae of the genus Symbiodinium form important symbioses within corals and other benthic marine animals. Dinoflagellates possess an extremely reduced plastid genome relative to those examined in plants and other algae. In dinoflagellates the plastid genes are located on small plasmids, commonly referred to as \\'minicircles\\'. However, the chloroplast genomes of dinoflagellates have only been extensively characterised from a handful of species. There is also evidence of considerable variation in the chloroplast genome organisation across those species that have been examined. We therefore characterised the chloroplast genome from an environmental coral isolate, in this case containing a symbiont belonging to the Symbiodinium sp. clade C3. The gene content of the genome is well conserved with respect to previously characterised genomes. However, unlike previously characterised dinoflagellate chloroplast genomes we did not identify any \\'empty\\' minicircles. The sequences of this chloroplast genome show a high rate of evolution relative to other algal species. Particularly notable was a surprisingly high level of sequence divergence within the core polypeptides of photosystem I, the reasons for which are currently unknown. This chloroplast genome also possesses distinctive codon usage and GC content. These features suggest that chloroplast genomes in Symbiodinium are highly plastic. © 2013 Adrian C. Barbrook.

  3. Investigating cytoskeletal function in chloroplast protrusion formation in the arctic-alpine plant Oxyria digyna.

    Science.gov (United States)

    Holzinger, A; Wasteneys, G O; Lütz, C

    2007-05-01

    Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria

  4. Complete sequencing of five araliaceae chloroplast genomes and the phylogenetic implications.

    Directory of Open Access Journals (Sweden)

    Rong Li

    Full Text Available BACKGROUND: The ginseng family (Araliaceae includes a number of economically important plant species. Previously phylogenetic studies circumscribed three major clades within the core ginseng plant family, yet the internal relationships of each major group have been poorly resolved perhaps due to rapid radiation of these lineages. Recent studies have shown that phyogenomics based on chloroplast genomes provides a viable way to resolve complex relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report the complete nucleotide sequences of five Araliaceae chloroplast genomes using next-generation sequencing technology. The five chloroplast genomes are 156,333-156,459 bp in length including a pair of inverted repeats (25,551-26,108 bp separated by the large single-copy (86,028-86,566 bp and small single-copy (18,021-19,117 bp regions. Each chloroplast genome contains the same 114 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 80 protein coding genes. Gene size, content, and order, AT content, and IR/SC boundary structure are similar among all Araliaceae chloroplast genomes. A total of 140 repeats were identified in the five chloroplast genomes with palindromic repeat as the most common type. Phylogenomic analyses using parsimony, likelihood, and Bayesian inference based on the complete chloroplast genomes strongly supported the monophyly of the Asian Palmate group and the Aralia-Panax group. Furthermore, the relationships among the sampled taxa within the Asian Palmate group were well resolved. Twenty-six DNA markers with the percentage of variable sites higher than 5% were identified, which may be useful for phylogenetic studies of Araliaceae. CONCLUSION: The chloroplast genomes of Araliaceae are highly conserved in all aspects of genome features. The large-scale phylogenomic data based on the complete chloroplast DNA sequences is shown to be effective for the phylogenetic reconstruction of Araliaceae.

  5. Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis

    Directory of Open Access Journals (Sweden)

    Zhou Xiangjun

    2011-11-01

    Full Text Available Abstract Background Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Results Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5 was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. Conclusions The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

  6. Acquired phototrophy through retention of functional chloroplasts increases growth efficiency of the sea slug Elysia viridis.

    Directory of Open Access Journals (Sweden)

    Finn A Baumgartner

    Full Text Available Photosynthesis is a fundamental process sustaining heterotrophic organisms at all trophic levels. Some mixotrophs can retain functional chloroplasts from food (kleptoplasty, and it is hypothesized that carbon acquired through kleptoplasty may enhance trophic energy transfer through increased host growth efficiency. Sacoglossan sea slugs are the only known metazoans capable of kleptoplasty, but the relative fitness contributions of heterotrophy through grazing, and phototrophy via kleptoplasts, are not well understood. Fitness benefits (i.e. increased survival or growth of kleptoplasty in sacoglossans are commonly studied in ecologically unrealistic conditions under extended periods of complete darkness and/or starvation. We compared the growth efficiency of the sacoglossan Elysia viridis with access to algal diets providing kleptoplasts of differing functionality under ecologically relevant light conditions. Individuals fed Codium fragile, which provide highly functional kleptoplasts, nearly doubled their growth efficiency under high compared to low light. In contrast, individuals fed Cladophora rupestris, which provided kleptoplasts of limited functionality, showed no difference in growth efficiency between light treatments. Slugs feeding on Codium, but not on Cladophora, showed higher relative electron transport rates (rETR in high compared to low light. Furthermore, there were no differences in the consumption rates of the slugs between different light treatments, and only small differences in nutritional traits of algal diets, indicating that the increased growth efficiency of E. viridis feeding on Codium was due to retention of functional kleptoplasts. Our results show that functional kleptoplasts from Codium can provide sacoglossan sea slugs with fitness advantages through photosynthesis.

  7. Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

    Institute of Scientific and Technical Information of China (English)

    XIAO Haidong; CHEN Changsheng; XU Yan; JI Dehua; XIE Chaotian

    2014-01-01

    Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplifica-tion of cDNA ends (RACE) technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, includ-ing a 5′untranslated region (UTR) of 92 bp, a 3′ UTR of 69 bp, and an open reading frame (ORF) of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR re-vealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

  8. Brain carbonic acid acidosis after acetazolamide

    DEFF Research Database (Denmark)

    Heuser, D; Astrup, J; Lassen, N A;

    1975-01-01

    of the carbonic anhydrase inhibition on the brain tissue. As a further support for this conclusion was considered the finding of a prolonged response time of brain pH to HCO3-minus i.v. to CO2-minus inhalation, and to hyperventilation after the acetazolamide inhibtion. No changes in brain extracelllular potassium......In cats in barbiturate anesthesia extracellular pH and potassium were continously recorded from brian cortex by implanted microelectrodes. Implantation of the electrodes preserved the low permeability of the blood-brain-barrier to HCO3-minus and H+ions as indicated by the development of brain...

  9. Increasing tomato fruit quality by enhancing fruit chloroplast function. A double-edged sword?

    Science.gov (United States)

    Cocaliadis, Maria Florencia; Fernández-Muñoz, Rafael; Pons, Clara; Orzaez, Diego; Granell, Antonio

    2014-08-01

    Fruits are generally regarded as photosynthate sinks as they rely on energy provided by sugars transported from leaves to carry out the highly demanding processes of development and ripening; eventually these imported photosynthates also contribute to the fruit organoleptic properties. Three recent reports have revealed, however, that transcriptional factors enhancing chloroplast development in fruit may result in higher contents not only of tomato fruit-specialized metabolites but also of sugars. In addition to suggesting new ways to improve fruit quality by fortifying fruit chloroplasts and plastids, these results prompted us to re-evaluate the importance of the contribution of chloroplasts/photosynthesis to fruit development and ripening.

  10. Functional chloroplasts in metazoan cells - a unique evolutionary strategy in animal life

    Directory of Open Access Journals (Sweden)

    Krug Patrick J

    2009-12-01

    Full Text Available Abstract Background Among metazoans, retention of functional diet-derived chloroplasts (kleptoplasty is known only from the sea slug taxon Sacoglossa (Gastropoda: Opisthobranchia. Intracellular maintenance of plastids in the slug's digestive epithelium has long attracted interest given its implications for understanding the evolution of endosymbiosis. However, photosynthetic ability varies widely among sacoglossans; some species have no plastid retention while others survive for months solely on photosynthesis. We present a molecular phylogenetic hypothesis for the Sacoglossa and a survey of kleptoplasty from representatives of all major clades. We sought to quantify variation in photosynthetic ability among lineages, identify phylogenetic origins of plastid retention, and assess whether kleptoplasty was a key character in the radiation of the Sacoglossa. Results Three levels of photosynthetic activity were detected: (1 no functional retention; (2 short-term retention lasting about one week; and (3 long-term retention for over a month. Phylogenetic analysis of one nuclear and two mitochondrial loci revealed reciprocal monophyly of the shelled Oxynoacea and shell-less Plakobranchacea, the latter comprising a monophyletic Plakobranchoidea and paraphyletic Limapontioidea. Only species in the Plakobranchoidea expressed short- or long-term kleptoplasty, most belonging to a speciose clade of slugs bearing parapodia (lateral flaps covering the dorsum. Bayesian ancestral character state reconstructions indicated that functional short-term retention arose once in the last common ancestor of Plakobranchoidea, and independently evolved into long-term retention in four derived species. Conclusion We propose a sequential progression from short- to long-term kleptoplasty, with different adaptations involved in each step. Short-term kleptoplasty likely arose as a deficiency in plastid digestion, yielding additional energy via the release of fixed carbon

  11. The complete chloroplast genome sequence of Anoectochilus emeiensis.

    Science.gov (United States)

    Zhu, Shuying; Niu, Zhitao; Yan, Wenjin; Xue, Qingyun; Ding, Xiaoyu

    2016-09-01

    The complete chloroplast (cp) genome sequence of Anoectochilus emeiensis, an extremely endangered medical plant with important economic value, was determined and characterized. The genome size was 152 650 bp, containing a pair of inverted repeats (IRs) (26 319 bp) which were separated by a large single copy (LSC) (82 670 bp) and a small single copy (SSC) (17 342 bp). The cpDNA of A. emeiensis contained 113 unique genes, including 79 protein coding genes, 30 tRNA genes and 4 rRNA genes. Among them, 18 genes contained one or two introns. The overall AT content of the genome was 63.1%. PMID:26403535

  12. Development of novel chloroplast microsatellite markers for Ginkgo biloba.

    Science.gov (United States)

    Xu, M; Xu, L A; Cao, F L; Zhang, H J; Yu, F X

    2015-07-13

    Ginkgo biloba is considered to be a living fossil that can be used to understand the ancient evolutionary history of gymnosperms, but little attention has been given to the study of its population genetics, molecular phylogeography, and genetic resources assessment. Chloroplast simple sequence repeat (cpSSR) markers are powerful tools for genetic studies of plants. In this study, a total of 30 perfect cpSSRs of Ginkgo were identified and characterized, including di-, tri, tetra-, penta-, and hexanucleotide repeats. Fifteen of 21 designed primer pairs were successfully amplified to yield specific polymerase chain reaction products from 16 Ginkgo cultivars. Polymorphic cpSSRs were further applied to determine the genetic variation of 116 individuals in 5 populations of G. biloba. The results showed that 24 and 76% genetic variation existed within and among populations of this species, respectively. These polymorphic and monomorphic cpSSR markers can be used to trace the origin and evolutionary history of Ginkgo.

  13. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    Energy Technology Data Exchange (ETDEWEB)

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  14. The complete chloroplast genomes of Cannabis sativa and Humulus lupulus.

    Science.gov (United States)

    Vergara, Daniela; White, Kristin H; Keepers, Kyle G; Kane, Nolan C

    2016-09-01

    Cannabis and Humulus are sister genera comprising the entirety of the Cannabaceae sensu stricto, including C. sativa L. (marijuana, hemp), and H. lupulus L. (hops) as two economically important crops. These two plants have been used by humans for many purposes including as a fiber, food, medicine, or inebriant in the case of C. sativa, and as a flavoring component in beer brewing in the case of H. lupulus. In this study, we report the complete chloroplast genomes for two distinct hemp varieties of C. sativa, Italian "Carmagnola" and Russian "Dagestani", and one Czech variety of H. lupulus "Saazer". Both C. sativa genomes are 153 871 bp in length, while the H. lupulus genome is 153 751 bp. The genomes from the two C. sativa varieties differ in 16 single nucleotide polymorphisms (SNPs), while the H. lupulus genome differs in 1722 SNPs from both C. sativa cultivars. PMID:26329384

  15. The complete chloroplast genomes of Cannabis sativa and Humulus lupulus.

    Science.gov (United States)

    Vergara, Daniela; White, Kristin H; Keepers, Kyle G; Kane, Nolan C

    2016-09-01

    Cannabis and Humulus are sister genera comprising the entirety of the Cannabaceae sensu stricto, including C. sativa L. (marijuana, hemp), and H. lupulus L. (hops) as two economically important crops. These two plants have been used by humans for many purposes including as a fiber, food, medicine, or inebriant in the case of C. sativa, and as a flavoring component in beer brewing in the case of H. lupulus. In this study, we report the complete chloroplast genomes for two distinct hemp varieties of C. sativa, Italian "Carmagnola" and Russian "Dagestani", and one Czech variety of H. lupulus "Saazer". Both C. sativa genomes are 153 871 bp in length, while the H. lupulus genome is 153 751 bp. The genomes from the two C. sativa varieties differ in 16 single nucleotide polymorphisms (SNPs), while the H. lupulus genome differs in 1722 SNPs from both C. sativa cultivars.

  16. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    Science.gov (United States)

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  17. The whole chloroplast genome of wild rice (Oryza australiensis).

    Science.gov (United States)

    Wu, Zhiqiang; Ge, Song

    2016-01-01

    The whole chloroplast genome of wild rice (Oryza australiensis) is characterized in this study. The genome size is 135,224  bp, exhibiting a typical circular structure including a pair of 25,776  bp inverted repeats (IRa,b) separated by a large single-copy region (LSC) of 82,212  bp and a small single-copy region (SSC) of 12,470  bp. The overall GC content of the genome is 38.95%. 110 unique genes were annotated, including 76 protein-coding genes, 4 ribosomal RNA genes, and 30t RNA genes. Among these, 18 are duplicated in the inverted repeat regions, 13 genes contain one intron, and 2 genes (rps12 and ycf3) have two introns.

  18. The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

    Science.gov (United States)

    Gurusamy, Raman; Lee, Do-Hyung; Park, SeonJoo

    2016-05-01

    The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns.

  19. The complete chloroplast genome sequence of Euonymus japonicus (Celastraceae).

    Science.gov (United States)

    Choi, Kyoung Su; Park, SeonJoo

    2016-09-01

    The complete chloroplast (cp) genome sequence of the Euonymus japonicus, the first sequenced of the genus Euonymus, was reported in this study. The total length was 157 637 bp, containing a pair of 26 678 bp inverted repeat region (IR), which were separated by small single copy (SSC) region and large single copy (LSC) region of 18 340 bp and 85 941 bp, respectively. This genome contains 107 unique genes, including 74 coding genes, four rRNA genes, and 29 tRNA genes. Seventeen genes contain intron of E. japonicus, of which three genes (clpP, ycf3, and rps12) include two introns. The maximum likelihood (ML) phylogenetic analysis revealed that E. japonicus was closely related to Manihot and Populus. PMID:26407184

  20. [Study of Chloroplast DNA Polymorphism in the Sunflower (Helianthus L.)].

    Science.gov (United States)

    Markina, N V; Usatov, A V; Logacheva, M D; Azarin, K V; Gorbachenko, C F; Kornienko, I V; Gavrilova, V A; Tihobaeva, V E

    2015-08-01

    The polymorphism of microsatellite loci of chloroplast genome in six Helianthus species and 46 lines of cultivated sunflower H. annuus (17 CMS lines and 29 Rf-lines) were studied. The differences between species are confined to four SSR loci. Within cultivated forms of the sunflower H. annuus, the polymorphism is absent. A comparative analysis was performed on sequences of the cpDNA inbred line 3629, line 398941 of the wild sunflower, and the American line HA383 H. annuus. As a result, 52 polymorphic loci represented by 27 SSR and 25 SNP were found; they can be used for genotyping of H. annuus samples, including cultural varieties: twelve polymorphic positions, of which eight are SSR and four are SNP. PMID:26601486