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Sample records for cerevisiae carbonic anhydrase

  1. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with anions.

    Science.gov (United States)

    Isik, Semra; Kockar, Feray; Arslan, Oktay; Guler, Ozen Ozensoy; Innocenti, Alessio; Supuran, Claudiu T

    2008-12-15

    The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically, and investigated for its inhibition with a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate, and some of its isosteric species. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5) s(-1) and k(cat)/K(m) of 9.8x10(7) M(-1) s(-1). scCA was weakly inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 16.8-55.6 mM) and strongly inhibited by bromide, iodide, and sulfamide (K(I)s of 8.7-10.8 microM). The other investigated anions showed inhibition constants in the low millimolar range.

  2. 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.

  3. Thermostable Carbonic Anhydrases in Biotechnological Applications

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    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.

  4. Coral Carbonic Anhydrases: Regulation by Ocean Acidification

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    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.

  5. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    Science.gov (United States)

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

    2016-06-03

    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.

  6. 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

  7. Genetics Home Reference: carbonic anhydrase VA deficiency

    Science.gov (United States)

    ... 19(3):199-229. Review. Citation on PubMed Shah GN, Rubbelke TS, Hendin J, Nguyen H, Waheed A, Shoemaker JD, Sly WS. Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose ...

  8. The Cellular Physiology of Carbonic Anhydrases

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    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.

  9. 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.

  10. Non-Classical Inhibition of Carbonic Anhydrase

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    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

  11. Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

    Science.gov (United States)

    Dixon, Daniel P; Van Ekeris, Leslie; Linser, Paul J

    2017-02-21

    In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the

  12. Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases

    Science.gov (United States)

    Dixon, Daniel P.; Van Ekeris, Leslie; Linser, Paul J.

    2017-01-01

    In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the

  13. 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.

  14. 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)

  15. 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.

  16. The role of carbonic anhydrase in hepatic glucose production.

    Science.gov (United States)

    Ismail, Ibrahim Salihu

    2016-12-14

    Considerable efforts are being made daily to discover novel therapeutic targets to better understand the mechanism for designing drugs in treating diabetes. Inhibition of hepatic gluconeogenesis by metformin remains the first line of oral therapy for managing type 2 diabetes. The link between rise in blood lactate level and reduction of hepatic glucose production with metformin usage remains to be determined. Carbonic anhydrase is proposed to be the link connecting blood lactate accumulation and inhibition of hepatic gluconeogenesis and thus could serve as a new therapeutic target for reducing hepatic glucose production. Understanding the link between rise in blood lactate level and the role of carbonic anhydrase in lactate uptake will be essential towards the development of a promising new antidiabetic medication.

  17. Future Perspective in Carbonic Anhydrase Inhibitors and its Drugs

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    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.

  18. Pilot absorption experiments with carbonic anhydrase enhanced MDEA

    DEFF Research Database (Denmark)

    Gladis, Arne; F. Lomholdt, Niels; Fosbøl, Philip Loldrup

    2017-01-01

    -methyl-diethanolamine (MDEA) solvent, with and without the enzyme carbonic anhydrase (CA). The absorption experiments were performed at atmospheric pressure and agas phase carbon dioxide mole fraction of 0.13. During experiments liquid samples were withdrawn at each meter of column height and the solvent loading...... was determined by both a density method and the BaCl2 method. After the solvent was loaded to equilibrium it was heated up and reintroduced into the column, where CO2 was stripped off using air as stripping gas. The addition of CA increased the mass transfer significantly in all experiments. Lower absorption...

  19. 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.

  20. Bioindication potential of carbonic anhydrase activity in anemones and corals.

    Science.gov (United States)

    Gilbert, A L; Guzmán, H M

    2001-09-01

    Activity levels of carbonic anhydrase (CA) were assessed in anemones Condylactis gigantea and Stichodactyla helianthus with laboratory exposures to copper, nickel, lead, and vanadium, and also in animals collected from polluted vs pristine field sites. CA activity was found to be decreased with increase in metal concentration and also in animals collected from the polluted field site. Preliminary assessments to adapt the CA assay for use in the widespread coral Montastraea cavernosa show decreased CA activity in specimens from the polluted field site and provide an avenue for future research aimed at more thoroughly describing coral CA activity for potential application in bioindication.

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

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    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.

  2. 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....

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

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    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.

  4. Carbonic anhydrase from Apis mellifera: purification and inhibition by pesticides.

    Science.gov (United States)

    Soydan, Ercan; Güler, Ahmet; Bıyık, Selim; Şentürk, Murat; Supuran, Claudiu T; Ekinci, Deniz

    2017-12-01

    Carbonic anhydrase (CA) enzymes have been shown to play an important role in ion transport and in pH regulation in several organisms. Despite this information and the wealth of knowledge regarding the significance of CA enzymes, few studies have been reported about bee CA enzymes and the hazardous effects of chemicals. Using Apis mellifera as a model, this study aimed to determine the risk of pesticides on Apis mellifera Carbonic anhydrase enzyme (Am CA). CA was initially purified from Apis mellifera spermatheca for the first time in the literature. The enzyme was purified with an overall purification of ∼35-fold with a molecular weight of ∼32 kDa. The enzyme was then exposed to pesticides, including tebuconazole, propoxur, carbaryl, carbofuran, simazine and atrazine. The six pesticides dose-dependently inhibited in vitro AmCA activity at low micromolar concentrations. IC50 values for the pesticides were 0.0030, 0.0321, 0.0031, 0.0087, 0.0273 and 0.0165 μM, respectively. The AmCA inhibition mechanism of these compounds is unknown at this moment.

  5. Building reactive copper centers in human carbonic anhydrase II.

    Science.gov (United States)

    Song, He; Weitz, Andrew C; Hendrich, Michael P; Lewis, Edwin A; Emerson, Joseph P

    2013-08-01

    Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu(2+) ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu(2+)-binding events have different affinities (K a approximately 5 × 10(12) and 1 × 10(10)), and both are enthalpically driven processes. Reconstituting these Cu(2+) sites under a range of conditions has allowed us to assign the Cu(2+)-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu(2+) sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

  6. Azobenzene-based inhibitors of human carbonic anhydrase II

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    Leander Simon Runtsch

    2015-07-01

    Full Text Available Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII. Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with Ki = 25–65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.

  7. A cytosolic carbonic anhydrase molecular switch occurs in the gills of metamorphic sea lamprey

    Science.gov (United States)

    Ferreira-Martins, D.; McCormick, Stephen; Campos, A.; Lopes-Marques, M.; Osorio, H.; Coimbra, J.; Castro, L.F.C.; Wilson, Jonthan M

    2016-01-01

    Carbonic anhydrase plays a key role in CO2 transport, acid-base and ion regulation and metabolic processes in vertebrates. While several carbonic anhydrase isoforms have been identified in numerous vertebrate species, basal lineages such as the cyclostomes have remained largely unexamined. Here we investigate the repertoire of cytoplasmic carbonic anhydrases in the sea lamprey (Petromyzon marinus), that has a complex life history marked by a dramatic metamorphosis from a benthic filter-feeding ammocoete larvae into a parasitic juvenile which migrates from freshwater to seawater. We have identified a novel carbonic anhydrase gene (ca19) beyond the single carbonic anhydrase gene (ca18) that was known previously. Phylogenetic analysis and synteny studies suggest that both carbonic anhydrase genes form one or two independent gene lineages and are most likely duplicates retained uniquely in cyclostomes. Quantitative PCR of ca19 and ca18 and protein expression in gill across metamorphosis show that the ca19 levels are highest in ammocoetes and decrease during metamorphosis while ca18 shows the opposite pattern with the highest levels in post-metamorphic juveniles. We propose that a unique molecular switch occurs during lamprey metamorphosis resulting in distinct gill carbonic anhydrases reflecting the contrasting life modes and habitats of these life-history stages.

  8. 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.

  9. The archetype gamma-class carbonic anhydrase (Cam) contains iron when synthesized in vivo.

    Science.gov (United States)

    Macauley, Sheridan R; Zimmerman, Sabrina A; Apolinario, Ethel E; Evilia, Caryn; Hou, Ya-Ming; Ferry, James G; Sowers, Kevin R

    2009-02-10

    A recombinant protein overproduction system was developed in Methanosarcina acetivorans to facilitate biochemical characterization of oxygen-sensitive metalloenzymes from strictly anaerobic species in the Archaea domain. The system was used to overproduce the archetype of the independently evolved gamma-class carbonic anhydrase. The overproduced enzyme was oxygen sensitive and had full incorporation of iron instead of zinc observed when overproduced in Escherichia coli. This, the first report of in vivo iron incorporation for any carbonic anhydrase, supports the need to reevaluate the role of iron in all classes of carbonic anhydrases derived from anaerobic environments.

  10. 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.

  11. 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.

  12. Natural Product Polyamines That Inhibit Human Carbonic Anhydrases

    Directory of Open Access Journals (Sweden)

    Rohan A. Davis

    2014-01-01

    Full Text Available Natural product compound collections have proven an effective way to access chemical diversity and recent findings have identified phenolic, coumarin, and polyamine natural products as atypical chemotypes that inhibit carbonic anhydrases (CAs. CA enzymes are implicated as targets of variable drug therapeutic classes and the discovery of selective, drug-like CA inhibitors is essential. Just two natural product polyamines, spermine and spermidine, have until now been investigated as CA inhibitors. In this study, five more complex natural product polyamines 1–5, derived from either marine sponge or fungi, were considered for inhibition of six different human CA isozymes of interest in therapeutic drug development. All compounds share a simple polyamine core fragment, either spermine or spermidine, yet display substantially different structure activity relationships for CA inhibition. Notably, polyamines 1–5 were submicromolar inhibitors of the cancer drug target CA IX, this is more potent than either spermine or spermidine.

  13. Characterization of human carbonic anhydrase III from skeletal muscle.

    Science.gov (United States)

    Carter, N; Jeffery, S; Shiels, A; Edwards, Y; Tipler, T; Hopkinson, D A

    1979-10-01

    A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.

  14. Generation of nitric oxide from nitrite by carbonic anhydrase

    DEFF Research Database (Denmark)

    Aamand, Rasmus; Dalsgaard, Thomas; Jensen, Frank B;

    2009-01-01

    In catalyzing the reversible hydration of CO2 to bicarbonate and protons, the ubiquitous enzyme carbonic anhydrase (CA) plays a crucial role in CO2 transport, in acid-base balance, and in linking local acidosis to O2 unloading from hemoglobin. Considering the structural similarity between...... in the reaction induces vasodilation in aortic rings. This reaction occurs under normoxic and hypoxic conditions and in various tissues at physiological levels of CA and nitrite. Furthermore, two specific inhibitors of the CO2 hydration, dorzolamide and acetazolamide, increase the CA-catalyzed production...... of vasoactive NO from nitrite. This enhancing effect may explain the known vasodilating effects of these drugs and indicates that CO2 and nitrite bind differently to the enzyme active site. Kinetic analyses show a higher reaction rate at high pH, suggesting that anionic nitrite participates more effectively...

  15. 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.

  16. 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....

  17. 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....

  18. The evolution of metazoan α-carbonic anhydrases and their roles in calcium carbonate biomineralization

    OpenAIRE

    Le Roy, Nathalie; Jackson, Daniel J.; Marie, Benjamin; Ramos-Silva, Paula; Marin, Frédéric

    2014-01-01

    The carbonic anhydrase (CA; EC 4.2.1.1) superfamily is a class of ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide. The ?-CA family, present in all metazoan clades, is a key enzyme involved in a wide range of physiological functions including pH regulation, respiration, photosynthesis, and biocalcification. This paper reviews the evolution of the ?-CA family, with an emphasis on metazoan ?-CA members involved in biocalcification. Phylogenetic analyses reveal...

  19. 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.

  20. Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes.

    Science.gov (United States)

    Arabaci, Betul; Gulcin, Ilhami; Alwasel, Saleh

    2014-07-10

    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.

  1. 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.

  2. 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.

  3. 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

  4. Molecular and biochemical analysis of the α class carbonic anhydrases in Caenorhabditis elegans.

    Science.gov (United States)

    Fasseas, Michael K; Tsikou, Daniela; Flemetakis, Emmanouil; Katinakis, Panagiotis

    2011-03-01

    In this study, in silico analysis of the Caenorhabditis elegans genome revealed six genes (cah-1, cah-2, cah-3, cah-4, cah-5, and cah-6) possibly encoding α class CAs (carbonic anhydrase). Real-time RT-PCR analysis revealed the temporal expression pattern of each gene, as well as changes in expression levels under different atmospheric conditions (stress). Cah-3 and cah-4 showed the highest levels of transcript accumulation, while most genes responded to the stress conditions. Yeast complementation showed that cah-3 was able to complement the function of Saccharomyces cerevisiae CA (NCE103) in vivo. Recombinant CAH-3, CAH-4a and CAH-5 enzymes, expressed in Escherichia coli were used for in vitro measurement of CA activity. However, in vitro activity was only detectable for CAH-4a. RNAi by feeding was performed on wild-type C. elegans for all genes. The worms were examined for a visible phenotype under normal and stress conditions (pH, CO(2)/O(2)). Silencing cah-3 and cah-4 may reduce the life-span of the worms (at 22 °C).

  5. 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.

  6. 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.

  7. 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.

  8. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    Directory of Open Access Journals (Sweden)

    Mariana Vieira Tomazett

    Full Text Available 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.

  9. 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...

  10. Carbonic Anhydrase Enhanced Carbon Capture: Kinetic Measurements and Pilot Plant Trials

    DEFF Research Database (Denmark)

    Gladis, Arne; Deslauriers, Maria Gundersen; Fosbøl, Philip Loldrup

    In this study the effect of carbonic anhydrase addition on the absorption of CO2 was investigated in a wetted wall column apparatus. Four different solvents: MEA (a primary amine), AMP (a sterically hindered primary amine), MDEA (a tertiary amine) and K2CO3 a carbonate salt solution were tested...... in concentrations from 5 to 50 wt%. Necessary mass transfer parameters such as liquid side mass transfer coefficient and solvent and enzyme reaction rates were determined in a temperature range from 298 to 328 K and benchmarked to a 30 wt% MEA solution. The study reveals that the addition of the enzyme carbonic...

  11. 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...

  12. 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.

  13. Carbonic anhydrase mimics for enhanced CO2 absorption in an amine-based capture solvent.

    Science.gov (United States)

    Kelsey, Rachael A; Miller, David A; Parkin, Sean R; Liu, Kun; Remias, Joe E; Yang, Yue; Lightstone, Felice C; Liu, Kunlei; Lippert, Cameron A; Odom, Susan A

    2016-01-07

    Two new small-molecule enzyme mimics of carbonic anhydrase were prepared and characterized. These complexes contain the salen-like ligand bis(hydroxyphenyl)phenanthroline. This ligand is similar to the salen-type ligands previously incorporated into carbonic anhydrase mimics but contains no hydrolyzable imine groups and therefore serves as a promising ligand scaffold for the synthesis of a more robust CO2 hydration catalyst. These homogeneous catalysts were investigated for CO2 hydration in concentrated primary amine solutions through which a dilute CO2 (14%) fluid stream was flowed and showed exceptional activity for increased CO2 absorption rates.

  14. 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.

  15. Antibody inhibiting enzymatic activity of tumour-associated carbonic anhydrase isoform IX

    NARCIS (Netherlands)

    Murri-Plesko, M.T.; Hulikova, A.; Oosterwijk, E.; Scott, A.M.; Zortea, A.; Harris, A.L.; Ritter, G.; Old, L.; Bauer, S.; Swietach, P.; Renner, C.

    2011-01-01

    Carbonic anhydrase IX (CAIX) is a hypoxia-induced, membrane-tethered enzyme that is highly expressed in many cancers. It catalyses the hydration of CO(2) to HCO(3)(-) and H(+), and the reverse dehydration reaction. Recent studies have shown an important role for CAIX in pH regulation and it has been

  16. 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 tensio...

  17. Structure and Metal Exchange in the Cadmium Carbonic anhydrase of Marine Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Xu,Y.; Feng, l.; Jeffrey, P.; Shi, Y.; Morel, F.

    2008-01-01

    Carbonic anhydrase, a zinc enzyme found in organisms from all kingdoms, catalyses the reversible hydration of carbon dioxide and is used for inorganic carbon acquisition by phytoplankton. In the oceans, where zinc is nearly depleted, diatoms use cadmium as a catalytic metal atom in cadmium carbonic anhydrase (CDCA). Here we report the crystal structures of CDCA in four distinct forms: cadmium-bound, zinc-bound, metal-free and acetate-bound. Despite lack of sequence homology, CDCA is a structural mimic of a functional {beta}-carbonic anhydrase dimer, with striking similarity in the spatial organization of the active site residues. CDCA readily exchanges cadmium and zinc at its active site--an apparently unique adaptation to oceanic life that is explained by a stable opening of the metal coordinating site in the absence of metal. Given the central role of diatoms in exporting carbon to the deep sea, their use of cadmium in an enzyme critical for carbon acquisition establishes a remarkable link between the global cycles of cadmium and carbon.

  18. 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.

  19. 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.

  20. 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

  1. Co-production of carbonic anhydrase and phycobiliproteins by Spirulina sp. and Synechococcus nidulans.

    Science.gov (United States)

    Ores, Joana da Costa; Amarante, Marina Campos Assumpção de; Kalil, Susana Juliano

    2016-11-01

    The aim of this work was to study the co-production of the carbonic anhydrase, C-phycocyanin and allophycocyanin during cyanobacteria growth. Spirulina sp. LEB 18 demonstrated a high potential for simultaneously obtaining the three products, achieving a carbonic anhydrase (CA) productivity of 0.97U/L/d and the highest C-phycocyanin (PC, 5.9μg/mL/d) and allophycocyanin (APC, 4.3μg/mL/d) productivities. In the extraction study, high extraction yields were obtained from Spirulina using an ultrasonic homogenizer (CA: 25.5U/g; PC: 90mg/g; APC: 70mg/g). From the same biomass, it was possible to obtain three biomolecules that present high industrial value.

  2. 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.

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

    Institute of Scientific and Technical Information of China (English)

    HUANG Huo-Qiang; PAN Xu-Lin; JI Chang-Jiu; ZENG Guang-Zhi; JIANG Li-Hua; FU Xiang; LIU Ji-Kai; HAO Xiao-Jiang; ZHANG Ying-Jun; TAN Ning-Hua

    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 CA Ⅱ, inhibition assay of carbonic anhydrase Ⅱ was performed, by which seven natural phenolic compounds, including four phenolics (grifolln, 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.

  4. 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.

  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. 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.

  7. Influence of temperature and solvent concentration on the kinetics of the enzyme carbonic anhydrase in carbon capture technology

    DEFF Research Database (Denmark)

    Gladis, Arne; Deslauriers, Maria Gundersen; Fosbøl, Philip Loldrup

    2017-01-01

    In this study the effect of carbonic anhydrase addition on the absorption of CO2 was investigated in a wetted wall column apparatus. Four different solvents: the primary amine monoethanolamine (MEA), the sterically hindered primary amine 2-amino-2-methyl-1-propanol (AMP), the tertiary amine N......-methyl-diethanolamine (MDEA) and the carbonate salt solution K2CO3 were compared in concentrations from 5 to 50 wt% in a temperature range of 298–328 K with and without enzyme. Necessary mass transfer parameters such as liquid side mass transfer coefficient and solvent and enzyme reaction rates were determined...... and benchmarked to a 30 wt% MEA solution. The study reveals that the addition of the enzyme carbonic anhydrase (CA) dramatically increases the liquid side mass transfer coefficient for MDEA, and K2CO3; AMP has a moderate increase whereas MEA was unchanged. The results confirm that just bicarbonate forming systems...

  8. 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.

  9. Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

    Directory of Open Access Journals (Sweden)

    Alvarez Bernardo V

    2013-01-01

    Full Text Available Abstract Background Carbonic anhydrase enzymes (CA catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide, a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. Methods We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14, or heart transplantation (HT, n = 13. CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. Results Expression of atrial and brain natriuretic peptide (ANP and BNP were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. Conclusions These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac

  10. 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.

  11. 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.

  12. 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.

  13. Saccharin: a lead compound for structure-based drug design of carbonic anhydrase IX inhibitors.

    Science.gov (United States)

    Mahon, Brian P; Hendon, Alex M; Driscoll, Jenna M; Rankin, Gregory M; Poulsen, Sally-Ann; Supuran, Claudiu T; McKenna, Robert

    2015-02-15

    Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

  14. 4-Amino-substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases

    Directory of Open Access Journals (Sweden)

    Kęstutis Rutkauskas

    2014-10-01

    Full Text Available A series of N-aryl-β-alanine derivatives and diazobenzenesulfonamides containing aliphatic rings were designed, synthesized, and their binding to carbonic anhydrases (CA I, II, VI, VII, XII, and XIII was studied by the fluorescent thermal shift assay and isothermal titration calorimetry. The results showed that 4-substituted diazobenzenesulfonamides were more potent CA binders than N-aryl-β-alanine derivatives. Most of the N-aryl-β-alanine derivatives showed better affinity for CA II while diazobenzenesulfonamides possessed nanomolar affinities towards CA I isozyme. X-ray crystallographic structures showed the modes of binding of both compound groups.

  15. The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis

    OpenAIRE

    Mahon, Brian P.; Bhatt, Avni; Socorro, Lilien; Driscoll, Jenna M.; Okoh, Cynthia; Lomelino, Carrie L.; Mboge, Mam Y.; Kurian, Justin J.; Tu, Chingkuang; Agbandje-McKenna, Mavis; Frost, Susan C; McKenna, Robert

    2016-01-01

    Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical prop...

  16. 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.

  17. 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.

  18. 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)

  19. Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter.

    Science.gov (United States)

    Becker, Holger M; Deitmer, Joachim W

    2007-05-04

    Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.

  20. 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.

  1. Carbonic anhydrase in calcified endoskeleton: novel activity in biocalcification in alcyonarian.

    Science.gov (United States)

    Rahman, M Azizur; Oomori, Tamotsu; Uehara, Tsuyoshi

    2008-01-01

    Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO(3) crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO(3) nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO(3) from a saturated solution containing CaCl(2) and NaHCO(3), indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.

  2. Synthesis of novel bisindolylmethanes: New carbonic anhydrase II inhibitors, docking, and 3D pharmacophore studies.

    Science.gov (United States)

    Imran, Syahrul; Taha, Muhammad; Ismail, Nor Hadiani; Fayyaz, Sharmeen; Khan, Khalid Mohammed; Choudhary, Muhammad Iqbal

    2016-10-01

    In this study, 45 bisindolylmethanes having sulfonamide moiety had been synthesized through 3 steps. In vitro assay for inhibition of carbonic anhydrase showed that some of the compounds having sulfonamide moiety are capable of inhibiting carbonic anhydrase II. Bisindoles having halogens at fifth position showed better inhibitory activity as compared to unsubstituted bisindoles. The results obtained from in vitro inhibitory activity were subjected through 3D QSAR and docking studies to identify important features contributing to the activity and further improve the structure. Pharmacophore studies suggest that bisindolylmethane moiety is contributing significantly towards the inhibition activity. Docking studies showed that compounds having nitro substituent (5g and 5i) were found to be able interact with Zn(2+) ion, Thr199, His94, His96, and His119, which interferes with the ZnOHThr199Glu106 hydrogen bond network. Bulky nitro substituent at ortho position for compound 5g prevents the compound from interacting with other residues like Thr199 and Thr200. Methyl substituent at ortho position for Compound 5i induces less steric hindrance effect, thus allowing second oxygen atom of sulfonamide to interact with Thr199 (2.51Å). Hydrogen bonding between NH on indole ring with Glu69 might have increased stability of ligand-receptor complex.

  3. Biochemistry and physiology of the β class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13.

    Science.gov (United States)

    Kumar, R Siva Sai; Hendrick, William; Correll, Jared B; Patterson, Andrew D; Melville, Stephen B; Ferry, James G

    2013-05-01

    The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the β class, the first β class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO₂ and 4.8 × 10⁶ s⁻¹ M⁻¹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO₂. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

  4. 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.

  5. Toxicity and Physiological Actions of Carbonic Anhydrase Inhibitors to Aedes aegypti and Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Sheena A. M. Francis

    2016-12-01

    Full Text Available The physiological role of carbonic anhydrases in pH and ion regulation is crucial to insect survival. We examined the toxic and neurophysiological effects of five carbonic anhydrase inhibitors (CAIs against Aedes aegypti. The 24 h larvicidal toxicities followed this rank order of potency: dichlorphenamide > methazolamide > acetazolamide = brinzolamide = dorzolamide. Larvicidal activity increased modestly in longer exposures, and affected larvae showed attenuated responses to probing without overt tremors, hyperexcitation, or convulsions. Acetazolamide and dichlorphenamide were toxic to adults when applied topically, but were of low potency and had an incomplete effect (<50% at 300 ng/mosquito even after injection. Dichlorphenamide was also the most toxic compound when fed to adult mosquitoes, and they displayed loss of posture and occasionally prolonged fluttering of the wings. Co-exposure with 500 ng of the synergist piperonyl butoxide (PBO increased the toxicity of dichlorphenamide ca. two-fold in feeding assays, indicating that low toxicity was not related to oxidative metabolism. Dichlorphenamide showed mild depolarizing and nerve discharge actions on insect neuromuscular and central nervous systems, respectively. These effects were increased in low buffer salines, indicating they were apparently related to loss of pH control in these tissues. Overall, sulfonamides displayed weak insecticidal properties on Aedes aegypti and are weak lead compounds.

  6. Extremely low-frequency electromagnetic fields affect lipid-linked carbonic anhydrase.

    Science.gov (United States)

    Ravera, Silvia; Pepe, Isidoro Mario; Calzia, Daniela; Morelli, Alessandro; Panfoli, Isabella

    2011-06-01

    In the last years, the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on the activity of different enzymes were investigated. Only the membrane-anchored enzymes did decrease their activity, up to 50%. In this work, the effect of ELF-EMF on bovine lung membrane carbonic anhydrase (CA) were studied. Carbonic anhydrases are a family of 14 zinc-containing isozymes catalyzing the reversible reaction: CO(2)+H(2)O = HCO(3)(- )+H(+). CA differ in catalytic activity and subcellular localization. CA IV, IX, XII, XIV, and XV are membrane bound. In particular, CA IV, which is expressed in the lung, is glycosyl phosphatidyl inositol-linked to the membrane, therefore it was a candidate to inhibition by ELF-EMF. Exposure to the membranes to a field of 75 Hz frequency and different amplitudes caused CA activity to a reproducible decrease in enzymatic activity by 17% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. When the source of enzyme was solubilized with Triton, the field lost its effect on CA enzymatic activity, suggesting a crucial role of the membrane, as well as of the particular linkage of the enzyme to it, in determining the conditions for CA inactivation. Results are discussed in terms of the possible physiologic effects of CA inhibition in target organs.

  7. Comparison of inhibition effects of some benzoic acid derivatives on sheep heart carbonic anhydrase

    Science.gov (United States)

    Kiliç, Deryanur; Yildiz, Melike; Şentürk, Murat; Erdoǧan, Orhan; Küfrevioǧlu, Ömer Irfan

    2016-04-01

    Carbonic anhydrase (CA) is a family of metalloenzymes that requires Zn as a cofactor and catalyze the quick conversion of CO2 to HCO3- and H+. Inhibitors of the carbonic anhydrases (CAs) have medical usage of significant diseases such as glaucoma, epilepsy, gastroduodenal ulcers, acid-base disequilibria and neurological disorders. In the present study, inhibition of CA with some benzoic derivatives (1-6) were investigated. Sheep heart CA (shCA) enzyme was isolated by means of designed affinity chromatography gel (cellulose-benzyl-sulfanylamide) 42.45-fold in a yield of 44 % with 564.65 EU/mg. Purified shCA enzyme was used in vitro studies. In the studies, IC50 values were calculated for 3-aminobenzoic acid (1), 4-aminobenzoic acid (2), 2-hydroxybenzoic acid (3), 2-benzoylbenzoic acid (4), 2,3-dimethoxybenzoic acid (5), and 3,4,5-trimethoxybenzoic acid (6), showing the inhibition effects on the purified enzyme. Such molecules can be used as pioneer for discovery of novel effective CA inhibitors for medicinal chemistry applications.

  8. [Carbonic anhydrase of blue-green alga Spirulina platensis].

    Science.gov (United States)

    Komarova, Iu M; Terekhova, I V; Doman, N G; Al'bitskaia, O N

    1976-01-01

    Carboanhydrase (carbonate-hydroliase EC 4.2.1.1.) is found in the extract of Spirulina platensis cells. A linear dependency of the enzyme activity on the protein concentration; pH optimum is found to be 8.0. Specific activity of carboanhydrase is 3 muM/min-mg of protein under the concentration of CO2 of 4-10(-3) M, appearing Michelis constant being 4.9-10(-3) M. The enzyme was stabilized with 10 mM of cisteine, its activity was inhibited by 50% with sulphanylamide (1-10(-5) M), acetazolamide (8--10(-7) M) and Cl- ions (5-10(-2) M). The activity of carboanhydrase, as well as the rate of NaH14CO3 fixation, depended on the pH value of cultural medium.

  9. Carbonic Anhydrase Inhibitors for the Treatment of Cystic Macular Lesions in Children With X-Linked Juvenile Retinoschisis

    NARCIS (Netherlands)

    Verbakel, S.K.; Ven, J.P.H. van de; Blanc, L.M.P. le; Groenewoud, J.M.M.; Jong, E.K.; Klevering, B.J.; Hoyng, C.B.

    2016-01-01

    Purpose: Little is known regarding the therapeutic effect of carbonic anhydrase inhibitors (CAIs) in the management of cystic macular lesions in children with X-linked juvenile retinoschisis (XLRS) despite the fact that this disease often manifests during childhood. Therefore, our goal was to determ

  10. Colocalization of carbonic anhydrase 9 expression and cell proliferation in human head and neck squamous cell carcinoma.

    NARCIS (Netherlands)

    Hoogsteen, I.J.; Marres, H.A.M.; Wijffels, K.I.E.M.; Rijken, P.F.J.W.; Peters, J.P.W.; Hoogen, F.J.A. van den; Oosterwijk, E.; Kogel, A.J. van der; Kaanders, J.H.A.M.

    2005-01-01

    PURPOSE: Tumor cells undergo a variety of biological changes under sustained hypoxic conditions, allowing cells to survive and retain their clonogenic potential. The purpose of this study is to relate the expression of the hypoxia marker carbonic anhydrase 9 (CA9) to the uptake of iododeoxyuridine (

  11. Synthesis, characterization and carbonic anhydrase inhibitory activity of novel benzothiazole derivatives.

    Science.gov (United States)

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

    2016-12-01

    N-protected amino acids were reacted with substituted benzothiazoles to give the corresponding N-protected amino acid-benzothiazole conjugates (60-89%). Their structures were confirmed by proton nuclear magnetic resonance ((1)H NMR), carbon-13 nuclear magnetic resonance ((13)C NMR), IR and elemental analysis. Their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activities were determined against two cytosolic human isoforms (hCA I and hCA II), one membrane-associated (hCA IV) and one transmembrane (hCA XII) enzyme by a stopped-flow CO2 hydrase assay method. The new compounds showed rather weak, micromolar inhibitory activity against most of these enzymes.

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

    Science.gov (United States)

    Moss, Adam M; Harris, Alon; Siesky, Brent; Rusia, Deepam; Williamson, Kathleen M; Shoshani, Yochai

    2010-04-26

    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.

  13. Carbonic anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic halves of the carbonic anhydrase active site.

    Science.gov (United States)

    Tanpure, Rajendra P; Ren, Bin; Peat, Thomas S; Bornaghi, Laurent F; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2015-02-12

    We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

  14. Carbonic anhydrase: a key regulatory and detoxifying enzyme for Karst plants.

    Science.gov (United States)

    Müller, Werner E G; Qiang, Li; Schröder, Heinz C; Hönig, Natalie; Yuan, Daoxian; Grebenjuk, Vlad A; Mussino, Francesca; Giovine, Marco; Wang, Xiaohong

    2014-01-01

    Karstification is a rapid process during which calcidic stones/limestones undergo dissolution with the consequence of a desertification of karst regions. A slow-down of those dissolution processes of Ca-carbonate can be approached by a reforestation program using karst-resistant plants that can resist alkaline pH and higher bicarbonate (HCO₃⁻) concentrations in the soil. Carbonic anhydrases (CA) are enzymes that mediate a rapid and reversible interconversion of CO₂ and HCO₃⁻. In the present study, the steady-state expression of a CA gene, encoding for the plant carbonic anhydrase from the parsley Petroselinum crispum, is monitored. The studies were primarily been performed during germination of the seeds up to the 12/14-day-old embryos. The CA cDNA was cloned. Quantitative polymerase chain reaction (qPCR) analysis revealed that the gene expression level of the P. crispum CA is strongly and significantly affected at more alkaline pH in the growth medium (pH 8.3). This abolishing effect is counteracted both by addition of HCO₃⁻ and by addition of polyphosphate (polyP) to the culture medium. In response to polyP, the increased pH in the vacuoles of the growing plants is normalized. The effect of polyP let us to propose that this polymer acts as a buffer system that facilitates the adjustment of the pH in the cytoplasm. In addition, it is proposed that polyP has the potential to act, especially in the karst, as a fertilizer that allows the karstic plants to cope with the adverse pH and HCO₃⁻ condition in the soil.

  15. Inhibition of carbonic anhydrase II by thioxolone: a mechanistic and structural study.

    Science.gov (United States)

    Barrese, Albert A; Genis, Caroli; Fisher, S Zoe; Orwenyo, Jared N; Kumara, Mudalige Thilak; Dutta, Subodh K; Phillips, Eric; Kiddle, James J; Tu, Chingkuang; Silverman, David N; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; McKenna, Robert; Tripp, Brian C

    2008-03-11

    This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791 . Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.

  16. Expression of the CHOP-inducible carbonic anhydrase CAVI-b is required for BDNF-mediated protection from hypoxia

    OpenAIRE

    Matthews, Tori A.; Abel, Allyssa; Demme, Chris; Sherman, Teresa; Pan, Pei-wen; Halterman, Marc W.; Parkkila, Seppo; Nehrke, Keith

    2013-01-01

    Carbonic anhydrases (CAs) comprise a family of zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide. CAs contribute to a myriad of physiological processes, including pH regulation, anion transport and water balance. To date, 16 known members of the mammalian alpha-CA family have been identified. Given that the catalytic family members share identical reaction chemistry, their physiologic roles are influenced greatly by their tissue and sub-cellular locations. CAVI ...

  17. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds.

    Science.gov (United States)

    Petrou, Anthi; Geronikaki, Athina; Terzi, Emine; Guler, Ozen Ozensoy; Tuccinardi, Tiziano; Supuran, Claudiu T

    2016-12-01

    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. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

  18. 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.

  19. 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.

  20. Agents described in the Molecular Imaging and Contrast Agent Database for imaging carbonic anhydrase IX expression.

    Science.gov (United States)

    Sneddon, Deborah; Poulsen, Sally-Ann

    2014-10-01

    Carbonic anhydrase IX (CA IX) is selectively expressed in a range of hypoxic tumours and is a validated endogenous hypoxia marker with prognostic significance; hence, CA IX is of great interest as a molecular imaging target in oncology. In this review, we present an overview of the different imaging agents and imaging modalities that have been applied for the in vivo detection of CA IX. The imaging agents reviewed are all entries in the Molecular Imaging and Contrast Agent Database (MICAD) and comprise antibody, antibody fragments and small molecule imaging agents. The effectiveness of these agents for imaging CA IX in vivo gave variable performance; however, a number of agents proved very capable. As molecular imaging has become indispensable in current medical practice we anticipate that the clinical significance of CA IX will see continued development and improvements in imaging agents for targeting this enzyme.

  1. Virtual screening of combinatorial library of novel benzenesulfonamides on mycobacterial carbonic anhydrase II

    Directory of Open Access Journals (Sweden)

    Dikant F.

    2016-12-01

    Full Text Available Combinatorial library of novel benzenesulfonamides was docked (Schrodinger Glide into mycobacterial carbonic anhydrase (mtCA II and human (hCA II isoforms with an aim to find drug candidates with selective activity on mtCA II. The predicted selectivity was calculated based on optimized MM-GBSA free energies for ligand enzyme interactions. Selectivity, LogP (o/w and interaction energy were used to calculate the selection index which determined the subset of best scoring molecules selected for further evaluation. Structure-activity relationship was found for fragment subsets, showing us the possible way regarding how to influence lipophilicity without affecting ligand-enzyme binding properties.

  2. 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.

  3. Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential.

    Science.gov (United States)

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2011-06-01

    Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn(2+)-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria.

  4. 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

  5. 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.

  6. Carbonic anhydrase inhibitors modify intracellular pH transients and contractions of rat middle cerebral arteries during CO2/HCO3(-) fluctuations.

    Science.gov (United States)

    Rasmussen, Jacob K; Boedtkjer, Ebbe

    2017-01-01

    The CO2/HCO3(-) buffer minimizes pH changes in response to acid-base loads, HCO3(-) provides substrate for Na(+),HCO3(-)-cotransporters and Cl(-)/HCO3(-)-exchangers, and H(+) and HCO3(-) modify vasomotor responses during acid-base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3(-) buffer. Switching from CO2/HCO3(-)-free to CO2/HCO3(-)-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3(-) uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na(+),HCO3(-)-cotransport and Na(+)/H(+)-exchange activity after NH4(+)-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3(-)-free conditions but absent when the buffer capacity and apparent H(+) mobility increase in the presence of CO2/HCO3(-) even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3(-) minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and

  7. Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite.

    Science.gov (United States)

    Chobanyan-Jürgens, Kristine; Schwarz, Alexandra; Böhmer, Anke; Beckmann, Bibiana; Gutzki, Frank-Mathias; Michaelsen, Jan T; Stichtenoth, Dirk O; Tsikas, Dimitrios

    2012-02-15

    Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.

  8. 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.

  9. Kinetic study of a novel thermo-stable α-carbonic anhydrase for biomimetic CO2 capture.

    Science.gov (United States)

    Russo, Maria Elena; Olivieri, Giuseppe; Capasso, Clemente; De Luca, Viviana; Marzocchella, Antonio; Salatino, Piero; Rossi, Mosè

    2013-09-10

    Biomimetic CO2 capture includes environmentally friendly solutions based on carbonic anhydrase (CA), an enzyme that increases CO2 absorption rate in conventional acid-gas scrubbing processes. The present contribution reports the characterization of a new recombinant carbonic anhydrase, SspCA, isolated from the thermophile bacterium Sulphurhydrogenibium yellowstonense sp. YO3AOP1. The kinetics of SspCA was characterized in terms of first order CO2 hydration rate according to a procedure based on CO2 absorption tests in a stirred cell apparatus. The first order kinetic constant at 25°C was 9.16 × 10(6) L/(mols). An appropriate investigation on SspCA stability was carried out to assess its long-term resistance to high temperatures as in all capture processes based on absorption/vacuum-desorption cycles. Its half-life was 53 and 8 days at 40 °C and 70 °C, respectively.

  10. Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity.

    OpenAIRE

    2008-01-01

    Abstract Background Acetaldehyde, the first metabolite of ethanol, can generate covalent modifications of proteins and cellular constituents. However, functional consequences of such modification remain poorly defined. In the present study, we examined acetaldehyde reaction with human carbonic anhydrase (CA) isozyme II, which has several features that make it a suitable target protein: It is widely expressed, its enzymatic activity can be monitored, its structural and catalytic properties are...

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

    OpenAIRE

    2013-01-01

    The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (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. Fibrobla...

  12. Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

    OpenAIRE

    Shah, Gul N.; Rubbelke, Timothy S.; Hendin, Joshua; Nguyen, Hien; Waheed, Abdul; Shoemaker, James D.; Sly, William S.

    2013-01-01

    Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium–potassium citrate-supplemented wate...

  13. A DFT-Based QSARs Study of Acetazolamide/Sulfanilamide Derivatives with Carbonic Anhydrase (CA-II Isozyme Inhibitory Activity

    Directory of Open Access Journals (Sweden)

    Oral Oltulu

    2007-02-01

    Full Text Available This study presents Quantitative Structure Activity Relationships (QSAR studyon a pool of 18 bio-active sulfonamide compounds which includes five acetazolamidederivatives, eight sulfanilamide derivatives and five clinically used sulfonamides moleculesas drugs namely acetazolamide, methazolamide, dichlorophenamide, ethoxolamide anddorzolamide. For all the compounds, initial geometry optimizations were carried out with amolecular mechanics (MM method using the MM force fields. The lowest energyconformations of the compounds obtained by the MM method were further optimized by theDensity Functional Theory (DFT method by employing Becke’s three-parameter hybridfunctional (B3LYP and 6-31G (d basis set. Molecular descriptors, dipole moment,electronegativity, total energy at 0 K, entropy at 298 K, HOMO and LUMO energiesobtained from DFT calculations provide valuable information and have a significant role inthe assessment of carbonic anhydrase (CA-II inhibitory activity of the compounds. By usingthe multiple linear regression technique several QSAR models have been drown up with thehelp these calculated descriptors and carbonic anhydrase (CA-II inhibitory data of themolecules. Among the obtained QSAR models presented in the study, statistically the mostsignificant one is a five parameters linear equation with the squared correlation coefficient R2 values of ca. 0.94 and the squared cross-validated correlation coefficient R2CV values of ca. 0.85. The results were discussed in the light of the main factors that influence theinhibitory activity of the carbonic anhydrase (CA-II isozyme.

  14. Allosteric reversion of Haemophilus influenzae β-carbonic anhydrase via a proline shift.

    Science.gov (United States)

    Hoffmann, Katherine M; Million-Perez, H Rachael; Merkhofer, Richard; Nicholson, Hilary; Rowlett, Roger S

    2015-01-20

    Haemophilus influenzae β-carbonic anhydrase (HICA) has been reverse-engineered in the allosteric site region to resemble the nonallosteric Pisum sativum enzyme in order to identify critical features of allostery and intersusbunit communication. Three variants (W39V/G41A, P48S/A49P, and W39V/G41A/P48S/A49P) were identified, through a comparison with a crystal structure of nonallosteric P. sativum β-carbonic anhydrase (PSCA, PDB 1EKJ ), to potentially revert HICA to a nonallosteric enzyme. The W39V/G41A and P48S/A49P mutations decreased the apparent kcat/Km proton dependence from 4 to 2 and 1, respectively, increasing the overall maximal kcat/Km to 16 ± 2 μM(-1) s(-1) (380% of wild type) and 17 ± 3 μM(-1) s(-1) (405% of wild type). The pKa values of the metal-bound water molecule based on the pH-rate profile kinetics (8.32 ± 0.04 for W39V/G41A and 8.3 ± 0.1 for P48S/A49P) were also slightly higher than that for the wild-type enzyme (7.74 ± 0.04). The P48S/A49P variant has lost all pH-rate cooperativity. The W39V/G41A/P48S/A49P variant's kinetics were unusual and were fit with a log-linear function with a slope 0.9 ± 0.2. The crystal structure of the W39V/G41A variant revealed an active site very similar to the T-state wild-type oligomer with bicarbonate trapped in the escort site. By contrast, the X-ray crystal structure of a proline shift variant (P48S/A49P) reveals that it has adopted an active site conformation nearly identical to that of nonallosteric β-carbonic anhydrase (R-state) for one chain, including a tight association with the dimer-exchanged N-terminal helices; the second chain in the asymmetric unit is associated in a biologically relevant oligomer, but it adopts a T-state conformation that is not capped by dimer-exchanged N-terminal helices. The hybrid R/T nature of HICA P48S/A49P structurally recapitulates the interruption of pH-rate cooperativity observed for this variant. Comparison of the conformations of the R and T chains of P48S/A49P

  15. How to get into bones: proton pump and carbonic anhydrase in Osedax boneworms.

    Science.gov (United States)

    Tresguerres, Martin; Katz, Sigrid; Rouse, Greg W

    2013-06-22

    Osedax are gutless siboglinid worms that thrive on vertebrate bones lying on the ocean floor, mainly those of whales. The posterior body of female Osedax penetrates into the bone forming extensions known as 'roots', which host heterotrophic symbiotic bacteria in bacteriocytes beneath the epidermis. The Osedax root epithelium presumably absorbs bone collagen and/or lipids, which are metabolized by the symbiotic bacteria that in turn serve for Osedax's nutrition. Here, we show that Osedax roots express extremely high amounts of vacuolar-H(+)-ATPase (VHA), which is located in the apical membrane and in cytoplasmic vesicles of root and ovisac epithelial cells. The enzyme carbonic anhydrase (CA), which catalyses the hydration of CO2 into H(+) and HCO3(-), is also expressed in roots and throughout Osedax body. These results suggest Osedax roots have massive acid-secreting capacity via VHA, fuelled by H(+) derived from the CA-catalysed hydration of CO2 produced by aerobic metabolism. We propose the secreted acid dissolves the bone carbonate matrix to then allow the absorption of bone-derived nutrients across the skin. In an exciting example of convergent evolution, this model for acid secretion is remarkably similar to mammalian osteoclast cells. However, while osteoclasts dissolve bone for repairing and remodelling, the Osedax root epithelium secretes acid to dissolve foreign bone to access nutrients.

  16. Characterization of urease and carbonic anhydrase producing bacteria and their role in calcite precipitation.

    Science.gov (United States)

    Achal, Varenyam; Pan, Xiangliang

    2011-03-01

    Urease and carbonic anhydrase (CA) are key enzymes in the chemical reaction of living organisms and have been found to be associated with calcification in a number of microorganisms and invertebrates. Three bacterial strains designated as AP4, AP6, and AP9 were isolated from highly alkaline soil samples using the enrichment culture technique. On the basis of various physiological tests and 16S rRNA sequence analysis, these three bacteria were identified as Bacillus sp., B. megaterium, and B. simplex. Further, these Bacillus species have been characterized for the production of urease and CA in the process of biocalcification. One of the isolates, AP6 produced 553 U/ml of urease and 5.61 EU/ml CA. All the strains were able to produce significant amount of exopolymeric substances and biofilm. Further, efficacy of these strains was tested for calcite production ability and results were correlated with urease and CA. Isolate AP6 precipitated 2.26 mg calcite/cell dry mass (mg). Our observations strongly suggest that it is not only urease but CA also plays an important role in microbially induced calcium carbonate precipitation process. The current work demonstrates that urease and CA producing microbes can be utilized in biocalcification as a sealing agent for filling the gaps or cracks and fissures in constructed facilities and natural formations alike.

  17. Carbonic anhydrase promotes the absorption rate of CO2 in post-combustion processes.

    Science.gov (United States)

    Vinoba, Mari; Bhagiyalakshmi, Margandan; Grace, Andrews Nirmala; Kim, Dae Hoon; Yoon, Yeoil; Nam, Sung Chan; Baek, Il Hyun; Jeong, Soon Kwan

    2013-05-09

    The rate of carbon dioxide (CO2) absorption by monoethanol amine (MEA), diethanol amine (DEA), N-methyl-2,2'-iminodiethanol (MDEA), and 2-amino-2-methyl 1-propanol (AMP) solutions was found to be enhanced by the addition of bovine carbonic anhydrase (CA), has been investigated using a vapor-liquid equilibrium (VLE) device. The enthalpy (-ΔHabs) of CO2 absorption and the absorption capacities of aqueous amines were measured in the presence and/or absence of CA enzyme via differential reaction calorimeter (DRC). The reaction temperature (ΔT) under adiabatic conditions was determined based on the DRC analysis. Bicarbonate and carbamate species formation mechanisms were elucidated by (1)H and (13)C NMR spectral analysis. The overall CO2 absorption rate (flux) and rate constant (kapp) followed the order MEA > DEA > AMP > MDEA in the absence or presence of CA. Hydration of CO2 by MDEA in the presence of CA directly produced bicarbonate, whereas AMP produced unstable carbamate intermediate, then underwent hydrolytic reaction and converted to bicarbonate. The MDEA > AMP > DEA > MEA reverse ordering of the enhanced CO2 flux and kapp in the presence of CA was due to bicarbonate formation by the tertiary and sterically hindered amines. Thus, CA increased the rate of CO2 absorption by MDEA by a factor of 3 relative to the rate of absorption by MDEA alone. The thermal effects suggested that CA yielded a higher activity at 40 °C.

  18. Design and simulation of rate-based CO2 capture processes using carbonic anhydrase (CA) applied to biogas

    DEFF Research Database (Denmark)

    Fosbøl, Philip Loldrup; Gaspar, Jozsef; Jacobsen, Bjartur

    2017-01-01

    a potential to create negative emissions using bio-energy carbon capture and storage (BECCS). All sectors are still in the need for applying more sustainable carbon capture and storage (CCS) technologies which result in lower energy consumption while reducing the impact on the environment. Recently several....... The advantage is a noticeably lower regeneration energy compared to primary and secondary amines. As a result the cost for stripping is significantly lower. Reactivated slow tertiary amines are applied in this study with the aim of reducing energy consumption. This is achieved byusing carbonic anhydrase (CA...

  19. Fluoroalkyl and alkyl chains have similar hydrophobicities in binding to the "hydrophobic wall" of carbonic anhydrase.

    Science.gov (United States)

    Mecinović, Jasmin; Snyder, Phillip W; Mirica, Katherine A; Bai, Serena; Mack, Eric T; Kwant, Richard L; Moustakas, Demetri T; Héroux, Annie; Whitesides, George M

    2011-09-07

    The hydrophobic effect, the free-energetically favorable association of nonpolar solutes in water, makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different but structurally similar hydrophobic groups, aliphatic hydrocarbons and aliphatic fluorocarbons, and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H(2)NSO(2)C(6)H(4)-CONHCH(2)(CX(2))(n)CX(3), n = 0-4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of nonoptimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and entropic contributions to the

  20. 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.

  1. 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.

  2. Molecular and biochemical analysis of the beta class carbonic anhydrases in Caenorhabditis elegans.

    Science.gov (United States)

    Fasseas, Michael K; Tsikou, Daniela; Flemetakis, Emmanouil; Katinakis, Panagiotis

    2010-07-01

    The beta class of the carbonic anhydrase (CA) enzyme family has been found in plants, yeast, bacteria and algae, but not in animals. Also, little is known concerning the CAs of C. elegans. Genes possibly encoding beta-CAs were revealed by in silico analysis of the C. elegans genome. Amino acid sequence and 3D structure analysis revealed a resemblance to both plant and cab-type beta-CAs. Temporal expression patterns of the two genes, as well as changes in expression levels under different atmospheric conditions (stress) were analyzed by real-time RT-PCR. Recombinant enzymes, expressed in E. coli were used for in vitro measurement of CA activity, while a yeast complementation experiment was performed in order to assess their ability to complement the function of S. crevisieae beta-CA (NCE103) in vivo. RNAi by feeding was performed on wild-type populations that were then examined for a visible phenotype under normal or various stress conditions (pH, CO(2)/O(2)). Two genes possibly encoding beta-CAs were revealed (bca-1 and y116a8c.28). Their products contain elements of both plant and cab-type CAs. Both assays showed that Y116a8c.28 is an active CA. Both genes showed significant levels of transcript accumulation during development, while they also responded to the stress conditions. No visible phenotype was scored under normal or stress conditions.

  3. 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.

  4. Oxygen Regulation of a Nodule-Located Carbonic Anhydrase in Alfalfa1

    Science.gov (United States)

    Gálvez, Susana; Hirsch, Ann M.; Wycoff, Keith L.; Hunt, Stephen; Layzell, David B.; Kondorosi, Adam; Crespi, Martin

    2000-01-01

    Control of the permeability to oxygen is critical for the function of symbiotic nitrogen fixation in legume nodules. The inner cortex (IC) seems to be a primary site for this regulation. In alfalfa (Medicago sativa) nodules, expression of the Msca1 gene encoding a carbonic anhydrase (CA) was previously found to be restricted to the IC. We have now raised antibodies against recombinant Msca1 protein and used them, together with antibodies raised against potato leaf CA, to demonstrate the presence of two forms of CA in mature nodules. Each antibody recognizes a different CA isoform in nodule tissues. Immunolocalization revealed that leaf-related CAs were localized primarily in the nitrogen-fixing zone, whereas the Msca1 protein was restricted exclusively to the IC region, in indeterminate and determinate nodules. In alfalfa nodules grown at various O2 concentrations, an inverse correlation was observed between the external oxygen pressure and Msca1 protein content in the IC, the site of the putative diffusion barrier. Thus Msca1 is a molecular target of physiological processes occurring in the IC cells involved in gas exchange in the nodule. PMID:11080283

  5. 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.

  6. 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.

  7. Antibodies reacting to carbonic anhydrase isozymes (I and II) and albumin in sera from dogs.

    Science.gov (United States)

    Nishita, Toshiho; Miyazaki, Rui; Miyazaki, Takae; Ochiai, Hideharu; Orito, Kensuke

    2016-06-01

    IgGs to carbonic anhydrase isozymes (CA-I and CA-II) and albumin were identified in dog serum. IgG titers were determined in the sera of asymptomatic dogs, and in dogs with atopic dermatitis, diarrhea and/or vomiting, diabetes and/or pancreatitis, kidney disease, hepatic disease, and thyroid gland disease, using ELISA. Low titres of IgG-reactive CA-I, CA-II, BSA, and CSA were found in the sera of healthy beagles. Compared with healthy beagles, there was a significant difference in the titers of antibodies against CA-I in asymptomatic dogs, dogs with diabetes and/or pancreatitis, or thyroid gland disease, or hepatic disease. Compared with healthy beagles, there was a significant difference in the antibody titer of anti-CA-II IgG in asymptomatic dogs and in those with hepatic disease. There was a significant difference in the antibody titer of anti-BSA IgG between healthy beagles and dogs with hepatic disease.

  8. Inhibition of the β-class carbonic anhydrases from Mycobacterium tuberculosis with carboxylic acids.

    Science.gov (United States)

    Maresca, Alfonso; Vullo, Daniela; Scozzafava, Andrea; Manole, Gheorghe; Supuran, Claudiu T

    2013-04-01

    The growth of Mycobacterium tuberculosis is strongly inhibited by weak acids although the mechanism by which these compounds act is not completely understood. A series of substituted benzoic acids, nipecotic acid, ortho- and para-coumaric acid, caffeic acid and ferulic acid were investigated as inhibitors of three β-class carbonic anhydrases (CAs, EC 4.2.1.1) from this pathogen, mtCA 1 (Rv1284), mtCA 2 (Rv3588c) and mtCA 3 (Rv3273). All three enzymes were inhibited with efficacies between the submicromolar to the micromolar one, depending on the scaffold present in the carboxylic acid. mtCA 3 was the isoform mostly inhibited by these compounds (K(I)s in the range of 0.11-0.97 µM); followed by mtCA 2 (K(I)s in the range of 0.59-8.10 µM), whereas against mtCA 1, these carboxylic acids showed inhibition constants in the range of 2.25-7.13 µM. This class of relatively underexplored β-CA inhibitors warrant further in vivo studies, as they may have the potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug or extensive multi-drug resistance.

  9. Genetic polymorphisms in the carbonic anhydrase VI gene and dental caries susceptibility.

    Science.gov (United States)

    Li, Z-Q; Hu, X-P; Zhou, J-Y; Xie, X-D; Zhang, J-M

    2015-06-01

    We investigated the role of 7 single nucleotide polymorphisms in the carbonic anhydrase (CA) VI gene (rs2274328, rs17032907, rs11576766, rs2274333, rs10864376, rs3765964, and rs6680186) and the possible association between these polymorphisms and dental caries susceptibility in a Northwestern Chinese population. We collected samples from 164 high caries experience and 191 very low caries experience and conducted a case-control study according to the number of decayed, missing, and filled teeth index and genotyped the 7 polymorphisms using a 384-well plate format with the Sequenom MassARRAY platform. Individuals carrying the rs17032907 TT genotype were more likely to have an increased risk of dental caries compared with carriers of the C/C genotype in the co-dominant model, with an odds ratio (95% confidence interval) of 2.144 (1.096-4.195). We also found that the haplotype (ACA) (rs2274328, rs17032907 and rs11576766) was associated with a low number of decayed, missing, and filled teeth index with an odds ratio (95% confidence interval) of 0.635 (0.440-0.918). However, we found no association between dental caries susceptibility and the rs2274328, rs11576766, rs2274333, rs10864376, rs3765964, and rs6680186 polymorphisms and other haplotypes. The rs17032907 genetic variant and the haplotype (ACA) of CA VI may be associated with dental caries susceptibility.

  10. Interaction of anions with a newly characterized alpha carbonic anhydrase from Halomonas sp.

    Science.gov (United States)

    Orhan, Furkan; Şentürk, Murat; Supuran, Claudiu T

    2016-12-01

    The inhibition and characterization of the α-class carbonic anhydrase (CA, EC 4.2.1.1) from the Halomonas sp. are reported for the first time. The enzyme was purified 91-fold with a yield of 39%, and a specific activity of 600 U/mg proteins was obtained. It has an optimum pH at 7.5, an optimum ionic strength at 20 mM and an optimum temperature at 20 °C. The following anions, SCN(-), Br(-), Cl(-), I(-), [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text] and [Formula: see text] showed inhibitory effects on the hydratase activity of the enzyme. Sulfate, sulfide, azide, nitrate, nitrite and iodide exhibited the strongest inhibitory activity, in the micromolar range (KI-s of 5.5-15.5 µM). SCN(-), Br(-), Cl(-), [Formula: see text] were moderate inhibitors, whereas other anions showed only weak activities. Our findings indicate that these anions inhibit the Halomonas sp. CA (HmCA) enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the Halomonas sp. and other organism CAs.

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

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    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.

  12. 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.

  13. Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration.

    Science.gov (United States)

    Yang, Zhenglin; Alvarez, Bernardo V; Chakarova, Christina; Jiang, Li; Karan, Goutam; Frederick, Jeanne M; Zhao, Yu; Sauvé, Yves; Li, Xi; Zrenner, Eberhart; Wissinger, Bernd; Hollander, Anneke I Den; Katz, Bradley; Baehr, Wolfgang; Cremers, Frans P; Casey, Joseph R; Bhattacharya, Shomi S; Zhang, Kang

    2005-01-15

    Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.

  14. 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.

  15. 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.

  16. 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.

  17. Effect of sulfonamides as carbonic anhydrase VA and VB inhibitors on mitochondrial metabolic energy conversion.

    Science.gov (United States)

    Arechederra, Robert L; Waheed, Abdul; Sly, William S; Supuran, Claudiu T; Minteer, Shelley D

    2013-03-15

    Obesity is quickly becoming an increasing problem in the developed world. One of the major fundamental causes of obesity and diabetes is mitochondria dysfunction due to faulty metabolic pathways which alter the metabolic substrate flux resulting in the development of these diseases. This paper examines the role of mitochondrial carbonic anhydrase (CA) isozymes in the metabolism of pyruvate, acetate, and succinate when specific isozyme inhibitors are present. Using a sensitive electrochemical approach of wired mitochondria to analytically measure metabolic energy conversion, we determine the resulting metabolic difference after addition of an inhibitory compound. We found that certain sulfonamide analogues displayed broad spectrum inhibition of metabolism, where others only had significant effect on some metabolic pathways. Pyruvate metabolism always displayed the most dramatically affected metabolism by the sulfonamides followed by fatty acid metabolism, and then finally succinate metabolism. This allows for the possibility of using designed sulfonamide analogues to target specific mitochondrial CA isozymes in order to subtly shift metabolism and glucogenesis flux to treat obesity and diabetes.

  18. SWATH-based proteomics identified carbonic anhydrase 2 as a potential diagnosis biomarker for nasopharyngeal carcinoma

    Science.gov (United States)

    Luo, Yanzhang; Mok, Tin Seak; Lin, Xiuxian; Zhang, Wanling; Cui, Yizhi; Guo, Jiahui; Chen, Xing; Zhang, Tao; Wang, Tong

    2017-01-01

    Nasopharyngeal carcinoma (NPC) is a serious threat to public health, and the biomarker discovery is of urgent needs. The data-independent mode (DIA) based sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry (MS) has been proved to be precise in protein quantitation and efficient for cancer biomarker researches. In this study, we performed the first SWATH-MS analysis comparing the NPC and normal tissues. Spike-in stable isotope labeling by amino acids in cell culture (super-SILAC) MS was used as a shotgun reference. We identified and quantified 1414 proteins across all SWATH-MS analyses. We found that SWATH-MS had a unique feature to preferentially detect proteins with smaller molecular weights than either super-SILAC MS or human proteome background. With SWATH-MS, 29 significant differentially express proteins (DEPs) were identified. Among them, carbonic anhydrase 2 (CA2) was selected for further validation per novelty, MS quality and other supporting rationale. With the tissue microarray analysis, we found that CA2 had an AUC of 0.94 in differentiating NPC from normal tissue samples. In conclusion, SWATH-MS has unique features in proteome analysis, and it leads to the identification of CA2 as a potentially new diagnostic biomarker for NPC. PMID:28117408

  19. Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII.

    Science.gov (United States)

    Ibrahim, Hany S; Abou-Seri, Sahar M; Tanc, Muhammet; Elaasser, Mahmoud M; Abdel-Aziz, Hatem A; Supuran, Claudiu T

    2015-10-20

    New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with KIs of 2.5-102 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively. Compounds 11e and 16e, with 5-NO2 substitution on the isatin ring, were found to be selective inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO2 group of both compounds participate in interactions with Asp132 within the hCA IX active site, and with residues Lys67 and Asp130 in hCA XII, respectively.

  20. 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.``

  1. 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

  2. Ultrastructural changes in the membrane system of isolated chloroplasts of spinach under the influence of carbonic anhydrase inhibitors AA and EA

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    Marina V. Vodka

    2013-04-01

    Full Text Available The effects of carbonic anhydrase inhibitors (АА and EA on the membrane system of isolated chloroplasts of spinach were investigated. Under the influence of AA the considerable alterations in granal structure occurred, the thickness of the granal thylakoids increased by 36% and the interspace between thylakoids by 10% comparable with the control. As a result of EA treatment, the thickness of granal thylakoids enhanced by 31% and the interspace between thylakoids increased by 8% in comparison to the control. It was shown that structure of the granal system of the chloroplast was more sensitive to AA than EA. The data obtained can indicate a decrease in the activity of the thylakoid carbonic anhydrase, inhibition of electron transport and photosynthetic process as a whole in the presence of carbonic anhydrase inhibitors (AA and EA.

  3. Structural basis of the oxidative activation of the carboxysomal [gamma]-carbonic anhydrase, CcmM

    Energy Technology Data Exchange (ETDEWEB)

    Peña, Kerry L.; Castel, Stephane E.; de Araujo, Charlotte; Espie, George S.; Kimber, Matthew S. (Guelph); (Toronto)

    2010-04-26

    Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO{sub 2} by carbonic anhydrase (CA) prior to fixation. Paradoxically, many {beta}-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal {beta}-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to {gamma}-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA - the first catalytically active bacterial {gamma}-CA reported. The 2.0 {angstrom} structure of CcmM209 reveals a trimeric, left-handed {beta}-helix structure that closely resembles Cam, except that residues 198-207 form a third {alpha}-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 {angstrom} structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the {beta}-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.

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

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    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.

  5. Molecular targeting of carbonic anhydrase IX in mice with hypoxic HT29 colorectal tumor xenografts.

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    Sean Carlin

    Full Text Available BACKGROUND: Carbonic anhydrase IX (CAIX is a membrane spanning protein involved in the enzymatic regulation of tumor acid-base balance. CAIX has been shown to be elevated in a number of hypoxic tumor types. The purpose of this study was to determine the efficiency of intact and IgG fragments of cG250 to target CAIX in vivo in a hypoxic tumor model. METHODOLOGY/PRINCIPAL FINDINGS: Conventional biodistribution studies were performed with (111In-DO3A-cG250, (111In-DO3A-F(ab'(2-cG250 and (111In-DO3A-Fab-cG250. Additional ex vivo analysis of the tumor was performed with markers for tumor hypoxia, blood perfusion and endogenous CAIX expression. All four data sets were digitally correlated to determine the optimal agent for determining hypoxia in a HT29 colon cancer xenograft. The HT29 human colorectal tumor xenografts show strong CAIX expression in hypoxic areas of poor blood perfusion. The intact IgG had an initial high focal uptake at the periphery of these hypoxic regions and penetration into the areas of highest CAIX expression over the 7-day study period. The lower molecular weight antibody fragments had a faster uptake into areas of high CAIX expression, but had a much lower absolute uptake at the optimal imaging times. CONCLUSIONS/SIGNIFICANCE: For the clinical detection of hypoxia induced CAIX using cG250 antibody based agents, imaging with the intact IgG at 7 days post injection would allow for the most sensitive and accurate detection of CAIX.

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

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    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

  7. Expression of carbonic anhydrase IX (CAIX) in malignant mesothelioma. An immunohistochemical and immunocytochemical study.

    Science.gov (United States)

    Capkova, L; Koubkova, L; Kodet, R

    2014-01-01

    Malignant mesothelioma is an aggressive tumor with a poor prognosis. Carbonic anhydrase IX (CAIX) is a membranously located metalloenzyme involved in pH homeostasis with influence on regulation of cell proliferation, oncogenesis and tumor progression. Much attention has been paid recently to carboanhydrases and their inhibitors as they offer an opportunity for both developing novel anticancer drugs, as well as diagnostic and prognostic tools. This study was designed to assess the expression of CAIX in malignant pleural and peritoneal mesotheliomas, their benign counterparts, and in pleural effusions from patients with malignant mesothelioma, metastatic carcinoma or a benign disease. Tissue blocks from 51 malignant mesotheliomas of pleura (47 cases; 41 epithelioid, 2 biphasic, 4 sarcomatoid) and peritoneum (4 cases; all epithelioid), 14 cases with normal or reactive pleural tissue, and 19 cell blocks were analyzed. CAIX expression was determined using immunohistochemistry and its membranous immunoreactivity was semiquantitatively evaluated. Specimens were divided into five subgroups according to the staining pattern and intensity.Overall, 92.2% (47/51) of mesotheliomas expressed CAIX. All epithelioid mesotheliomas showed CAIX positivity, which was predominantly strong and diffuse (73.3%, 33/45). Sarcomatoid mesotheliomas and sarcomatoid areas in biphasic mesotheliomas were negative. A strong diffuse staining was observed in all cases of normal mesothelia. In pleural effusions, CAIX expression was observed in malignant cells as well as in benign mesothelial cells. In conclusion, CAIX is expressed virtually in all mesotheliomas except for sarcomatoid subtype, and in benign mesothelia. There are probably more mechanisms of CAIX overexpression than hypoxia-induced in malignant mesothelioma, with the influence of other tissue specific transcription or growth factors depending on the type of the cell lineage. CAIX immunoreactivity is not a reliable diagnostic marker for

  8. 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.

  9. Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

    Science.gov (United States)

    Rudenko, Natalia N; Ignatova, Lyudmila K; Ivanov, Boris N

    2007-01-01

    Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids.

  10. Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

    Institute of Scientific and Technical Information of China (English)

    CHAI Yurong; LU Yumin; WANG Tianyun; HOU Weihong; XUE Lexun

    2006-01-01

    Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficientpromoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase (CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics.Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

  11. Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII.

    Science.gov (United States)

    Ignatova, Lyudmila K; Rudenko, Natalia N; Mudrik, Vilen A; Fedorchuk, Tat'yana P; Ivanov, Boris N

    2011-12-01

    The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg(2+) in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10(-7) M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10(-9) M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10(-7) M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants.

  12. The ctnG gene encodes carbonic anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aurantiacus.

    Science.gov (United States)

    Li, Yan-Ping; Tang, Xiao; Wu, Wei; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2015-01-01

    Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination.

  13. Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation▿

    OpenAIRE

    2009-01-01

    Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically character...

  14. A review of the pharmacology of carbonic anhydrase inhibitors for the treatment of glaucoma in dogs and cats.

    Science.gov (United States)

    Maślanka, Tomasz

    2015-03-01

    Glaucoma is a heterogeneous group of disorders usually associated with elevated intraocular pressure (IOP), leading to optic nerve damage, retinal ganglion cell death and irreversible vision loss. Therefore, medications that lower IOP are the mainstay of glaucoma therapy. Carbonic anhydrase inhibitors (CAIs) are some of the principal drugs used in the management of canine and feline glaucoma. This paper summarises current knowledge of the mechanism of action of these agents and their effect on IOP in dogs and cats. It also discusses potential harmful side effects of CAIs and presents current opinions about their role and place in the medical management of glaucoma in small animals.

  15. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

    Science.gov (United States)

    Andersson, Marlene; Chen, Gefei; Otikovs, Martins; Landreh, Michael; Nordling, Kerstin; Kronqvist, Nina; Westermark, Per; Jörnvall, Hans; Knight, Stefan; Ridderstråle, Yvonne; Holm, Lena; Meng, Qing; Jaudzems, Kristaps; Chesler, Mitchell; Johansson, Jan; Rising, Anna

    2014-08-01

    Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

  16. Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

    Directory of Open Access Journals (Sweden)

    Marlene Andersson

    2014-08-01

    Full Text Available Spider silk fibers are produced from soluble proteins (spidroins under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2 in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

  17. Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana.

    Science.gov (United States)

    Tachibana, Masaaki; Allen, Andrew E; Kikutani, Sae; Endo, Yuri; Bowler, Chris; Matsuda, Yusuke

    2011-09-01

    It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO(2) conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO(2) and 5% CO(2). However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO(2). Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm

  18. 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

  19. Carbonic Anhydrase I Is Recognized by an SOD1 Antibody upon Biotinylation of Human Spinal Cord Extracts

    Directory of Open Access Journals (Sweden)

    Robert Bowser

    2010-10-01

    Full Text Available We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1 antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS. This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I. Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.

  20. 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.

  1. The structural comparison between membrane-associated human carbonic anhydrases provides insights into drug design of selective inhibitors.

    Science.gov (United States)

    Alterio, Vincenzo; Pan, Peiwen; Parkkila, Seppo; Buonanno, Martina; Supuran, Claudiu T; Monti, Simona M; De Simone, Giuseppina

    2014-07-01

    Carbonic anhydrase isoform XIV (CA XIV) is the last member of the human (h) CA family discovered so far, being localized in brain, kidneys, colon, small intestine, urinary bladder, liver, and spinal cord. It has recently been described as a possible drug target for treatment of epilepsy, some retinopathies as well as some skin tumors. Human carbonic anhydrase (hCA) XIV is a membrane-associated protein consisting of an N-terminal extracellular domain, a putative transmembrane region, and a small cytoplasmic tail. In this article, we report the expression, purification, and the crystallographic structure of the entire extracellular domain of this enzyme. The analysis of the structure revealed the typical α-CA fold, in which a 10-stranded β-sheet forms the core of the molecule, while the comparison with all the other membrane associated isoforms (hCAs IV, IX, and XII) allowed to identify the diverse oligomeric arrangement and the sequence and structural differences observed in the region 127-136 as the main factors to consider in the design of selective inhibitors for each one of the membrane associated α-CAs.

  2. Evaluation of the therapeutic potential of carbonic anhydrase inhibitors in two animal models of dystrophin deficient muscular dystrophy.

    Science.gov (United States)

    Giacomotto, Jean; Pertl, Cordula; Borrel, Caroline; Walter, Maggie C; Bulst, Stefanie; Johnsen, Bob; Baillie, David L; Lochmüller, Hanns; Thirion, Christian; Ségalat, Laurent

    2009-11-01

    Duchenne Muscular Dystrophy is an inherited muscle degeneration disease for which there is still no efficient treatment. However, compounds active on the disease may already exist among approved drugs but are difficult to identify in the absence of cellular models. We used the Caenorhabditis elegans animal model to screen a collection of 1000 already approved compounds. Two of the most active hits obtained were methazolamide and dichlorphenamide, carbonic anhydrase inhibitors widely used in human therapy. In C. elegans, these drugs were shown to interact with CAH-4, a putative carbonic anhydrase. The therapeutic efficacy of these compounds was further validated in long-term experiments on mdx mice, the mouse model of Duchenne Muscular Dystrophy. Mice were treated for 120 days with food containing methazolamide or dichlorphenamide at two doses each. Musculus tibialis anterior and diaphragm muscles were histologically analyzed and isometric muscle force was measured in M. extensor digitorum longus. Both substances increased the tetanic muscle force in the treated M. extensor digitorum longus muscle group, dichlorphenamide increased the force significantly by 30%, but both drugs failed to increase resistance of muscle fibres to eccentric contractions. Histological analysis revealed a reduction of centrally nucleated fibers in M. tibialis anterior and diaphragm in the treated groups. These studies further demonstrated that a C. elegans-based screen coupled with a mouse model validation strategy can lead to the identification of potential pharmacological agents for rare diseases.

  3. 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.

  4. Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

    Science.gov (United States)

    Hall, G. E.; Kenny, A. D.

    1985-01-01

    The calvaria of 5-to-6-day-old mice treated with 1 x 10 to the -8th M of 1,25(OH)2D3 in vitro for 48 hours are examined in order to study the function of carbonic anhydrase in bone resorption. Calcium concentrations in the culture were measured to assess bone resorption. It is observed that 1,25(OH)2D3 effectively stimulates bone resorption in vitro and the resorption is dose-dependent. The effects of azetazolamide on 1,25(OH)2D3-induced bone resorption are investigated. The data reveal that 1,25(OH)2D3-induced calcium release is associated with an increase in the carbonic anhydrase activity of bone, and bone alkaline phosphatase activity is decreased and acid phosphatase activity is increased in response to 1,25(OH)2D3. A two-fold mechanism for 1,25(OH)2D3-induced bone resorption is proposed; the first mechanism is an indirect activation of osteoclasts and the second involves an interaction between hormone and osteoclast precursors.

  5. Inhibition of bacterial carbonic anhydrases and zinc proteases: from orphan targets to innovative new antibiotic drugs.

    Science.gov (United States)

    Supuran, C T

    2012-01-01

    Zinc-containing enzymes, such as carbonic anhydrases (CAs) and metalloproteases (MPs) play critical functions in bacteria, being involved in various steps of their life cycle, which are important for survival, colonization, acquisition of nutrients for growth and proliferation, facilitation of dissemination, invasion and pathogenicity. The development of resistance to many classes of clinically used antibiotics emphasizes the need of new antibacterial drug targets to be explored. There is a wealth of data regarding bacterial CAs and zinc MPs present in many pathogenic species, such as Neisseria spp., Helycobacter pylori Escherichia coli, Mycobacterium tuberculosis, Brucella spp., Streptococcus pneumoniae, Salmonella enterica, Haemophilus influenzae, Listeria spp, Vibrio spp., Pseudomonas aeruginosa, Legionella pneumophila, Streptomyces spp., Clostridium spp., Enterococcus spp., etc. Some of these enzymes have been cloned, purified and characterized by crystallographic techniques. However, for the moment, few potent and specific inhibitors for bacterial MPs have been reported except for Clostridium histolyticum collagenase, botulinum and tetanus neurotoxin and anthrax lethal factor, which will be reviewed in this article. Bacteria encode α-,β-, and/or γ-CA families, but up to now only the first two classes have been investigated in some detail in different species. The α-CAs from Neisseria spp. and H. pylori as well as the β-class enzymes from E. coli, H. pylori, M. tuberculosis, Brucella spp., S. pneumoniae, S. enterica and H. influenzae have been cloned and characterized. The catalytic/inhibition mechanisms of these CAs are well understood as X-ray crystal structures are available for some of them, but no adducts of these enzymes with inhibitors have been characterized so far. In vitro and in vivo studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates have been reported. Only for Neisseria spp., H. pylori, B. suis and S

  6. 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

  7. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

    Directory of Open Access Journals (Sweden)

    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  8. Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX-specific chimeric monoclonal antibody cG250

    NARCIS (Netherlands)

    Oosterwijk-Wakka, J.C.; Kats-Ugurlu, G.; Leenders, W.P.J.; Kiemeney, L.A.L.M.; Old, L.J.; Mulders, P.F.A.; Oosterwijk, E.

    2011-01-01

    OBJECTIVE: To investigate the effect of three different tyrosine kinase inhibitors (TKIs) on the biodistribution of chimeric monoclonal antibody (mAb) cG250, which identifies carbonic anhydrase IX (CAIX), in nude mice bearing human renal cell carcinoma (RCC) xenografts. TKIs represent the best, but

  9. Carbonic Anhydrase II: A Model System for Artificial Copper Center Design, Protein-guided Cycloadditions, Tethering Screenings and Fragment-based Lead Discovery

    OpenAIRE

    Schulze Wischeler, Johannes

    2010-01-01

    In this thesis a variety of quite different fragment-based lead discovery approaches have been applied to the target protein carbonic anhydrase II. The different projects were strongly supported and methodologically tailored towards protein crystallography; a method which is currently emerging as a routine analytical tool. This maturation mainly results from improved radiation sources and enhanced computing power. About 200-250 da...

  10. Rate-based modelling and validation of a pilot absorber using MDEA enhanced with carbonic anhydrase (CA)

    DEFF Research Database (Denmark)

    Gaspar, Jozsef; Gladis, Arne; Woodley, John

    2017-01-01

    The great paradox of the 21st century is that we must meet the increasing global demand for energy and products while simultaneously mitigating the climate change. If both these criteria are to be met, carbon capture and storage is an imperative technology for sustainable energy infrastructure...... development. Post-combustion capture is a mature capture technology, however, to make it economically attractive, design of innovative solvents and process optimization is of crucial importance. An example for promising solvent is MDEA enhanced with carbonic anhydrase (CA), due to its fast kinetics and low...... for different L/G ratios, lean CO2 loadings, gas CO2 content and packing height. We show that the developed model is suitable for CO2 capture simulation and optimization using MDEA and MDEA enhanced with CA. Furthermore, we investigate the accuracy of the General Method (GM) enhancement factor model for CO2...

  11. 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.

  12. Inhibition of carbonic anhydrase from Trypanosoma cruzi for the management of Chagas disease: an underexplored therapeutic opportunity.

    Science.gov (United States)

    Supuran, Claudiu T

    2016-01-01

    An α-carbonic anhydrases (CAs, EC 4.2.1.1) was recently discovered, cloned and characterized in the genome of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, a neglected but widespread tropical disease. Inhibition of this α-CAs (TcCA) with anions, sulfonamides, sulfamates, thiols and hydroxamates has been investigated in detail, with several low nanomolar in vitro inhibitors. Although the sulfonamides were the best in vitro inhibitors, they showed no ex vivo anti-T. cruzi activity, due to poor penetration. However, some thiols and hydroxamates acting as low nanomolar TcCA inhibitors also showed significant antitrypanosomal ex vivo activity, making this enzyme an attractive yet underexplored drug target for the management of Chagas disease.

  13. A class of sulfonamides with strong inhibitory action against the α-carbonic anhydrase from Trypanosoma cruzi.

    Science.gov (United States)

    Güzel-Akdemir, Özlen; Akdemir, Atilla; Pan, Peiwen; Vermelho, Alane B; Parkkila, Seppo; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2013-07-25

    Trypanosoma cruzi, the causative agent of Chagas disease, encodes for an α-carbonic anhydrase (CA, EC 4.2.1.1) possessing high catalytic activity (TcCA) which was recently characterized (Pan et al. J. Med. Chem. 2013, 56, 1761-1771). A new class of sulfonamides possessing low nanomolar/subnanomolar TcCA inhibitory activity is described here. Aromatic/heterocyclic sulfonamides incorporating halogeno/methoxyphenacetamido tails inhibited TcCA with KIs in the range of 0.5-12.5 nM, being less effective against the human off-target isoforms hCA I and II. A homology model of TcCA helped us to rationalize the excellent inhibition profile of these compounds against the protozoan enzyme, a putative new antitrypanosoma drug target. These compounds were ineffective antitrypanosomal agents in vivo due to penetrability problems of these highly polar molecules that possess sulfonamide moieties.

  14. High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design.

    Science.gov (United States)

    Sippel, Katherine H; Robbins, Arthur H; Domsic, John; Genis, Caroli; Agbandje-McKenna, Mavis; McKenna, Robert

    2009-10-01

    The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1 A resolution and refined to an R(cryst) of 11.2% and an R(free) of 14.7%. As observed in previous CA II-inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed.

  15. 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

  16. Identifying motor and sensory myelinated axons in rabbit peripheral nerves by histochemical staining for carbonic anhydrase and cholinesterase activities

    Science.gov (United States)

    Riley, Danny A.; Sanger, James R.; Matloub, Hani S.; Yousif, N. John; Bain, James L. W.

    1988-01-01

    Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myeliated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50 percent or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.

  17. Carbon anhydrase IX specific immune responses in patients with metastatic renal cell carcinoma potentially cured by interleukin-2 based immunotherapy

    DEFF Research Database (Denmark)

    Rasmussen, Susanne; Donskov, Frede; Pedersen, Johannes W

    2013-01-01

    Abstract The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell...... of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease...... interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent....

  18. Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

    Science.gov (United States)

    Papalia, Giuseppe A; Leavitt, Stephanie; Bynum, Maggie A; Katsamba, Phinikoula S; Wilton, Rosemarie; Qiu, Huawei; Steukers, Mieke; Wang, Siming; Bindu, Lakshman; Phogat, Sanjay; Giannetti, Anthony M; Ryan, Thomas E; Pudlak, Victoria A; Matusiewicz, Katarzyna; Michelson, Klaus M; Nowakowski, Agnes; Pham-Baginski, Anh; Brooks, Jonathan; Tieman, Bryan C; Bruce, Barry D; Vaughn, Michael; Baksh, Michael; Cho, Yun Hee; Wit, Mieke De; Smets, Alexandra; Vandersmissen, Johan; Michiels, Lieve; Myszka, David G

    2006-12-01

    In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.

  19. Effect the some heavy metals on carbonic anhydrase enzymes activities from non-tumour and tumour human stomach

    OpenAIRE

    2015-01-01

    In this study, in vitro effects of certain heavy metals on the human carbonic anhydrase enzyme were examined. Inhibitory effects of metal ions ( Pb2+, Cu2+, Fe2+,Cr2+, Al3+, Ni2+, Mn2+, Cd2+, Zn2+, and Mg2+) were observed in tumour and non-tumour tissue. IC50 values were calculated for metals. The Cu2+, Zn2+, Ni2+, Cd2+ and Mg2+ IC50 values of tumour tissue were calculated as 0.034mM, 0.426mM, 0.597mM, 0.878mM and 2.52mM respectively. The Cu2+, Zn2+, Ni2+, Cd2+ and Mg2+  IC50 values of non-tu...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Identifying potential selective fluorescent probes for cancer-associated protein carbonic anhydrase IX using a computational approach.

    Science.gov (United States)

    Kamstra, Rhiannon L; Floriano, Wely B

    2014-11-01

    Carbonic anhydrase IX (CAIX) is a biomarker for tumor hypoxia. Fluorescent inhibitors of CAIX have been used to study hypoxic tumor cell lines. However, these inhibitor-based fluorescent probes may have a therapeutic effect that is not appropriate for monitoring treatment efficacy. In the search for novel fluorescent probes that are not based on known inhibitors, a database of 20,860 fluorescent compounds was virtually screened against CAIX using hierarchical virtual ligand screening (HierVLS). The screening database contained 14,862 compounds tagged with the ATTO680 fluorophore plus an additional 5998 intrinsically fluorescent compounds. Overall ranking of compounds to identify hit molecular probe candidates utilized a principal component analysis (PCA) approach. Four potential binding sites, including the catalytic site, were identified within the structure of the protein and targeted for virtual screening. Available sequence information for 23 carbonic anhydrase isoforms was used to prioritize the four sites based on the estimated "uniqueness" of each site in CAIX relative to the other isoforms. A database of 32 known inhibitors and 478 decoy compounds was used to validate the methodology. A receiver-operating characteristic (ROC) analysis using the first principal component (PC1) as predictive score for the validation database yielded an area under the curve (AUC) of 0.92. AUC is interpreted as the probability that a binder will have a better score than a non-binder. The use of first component analysis of binding energies for multiple sites is a novel approach for hit selection. The very high prediction power for this approach increases confidence in the outcome from the fluorescent library screening. Ten of the top scoring candidates for isoform-selective putative binding sites are suggested for future testing as fluorescent molecular probe candidates.

  2. Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties.

    Science.gov (United States)

    Genis, Caroli; Sippel, Katherine H; Case, Nicolette; Cao, Wengang; Avvaru, Balendu Sankara; Tartaglia, Lawrence J; Govindasamy, Lakshmanan; Tu, Chingkuang; Agbandje-McKenna, Mavis; Silverman, David N; Rosser, Charles J; McKenna, Robert

    2009-02-17

    Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.

  3. Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

    Science.gov (United States)

    Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

    2017-02-01

    We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. The changes in the chloroplast membranes of pea leaves under the influence of carbonic anhydrase inhibitors (ions of copper and zinc

    Directory of Open Access Journals (Sweden)

    M.V. Vodka

    2014-04-01

    Full Text Available Тhe effects of carbonic anhydrase inhibitors, such as ions Cu2+ and Zn2+, on the membrane system of chloroplasts in pea leaves were investigated. After treatment of pea leaves with 250 mM Cu2+ or 400 mM Zn2+ we observed changes in the granal structure and compactness of the thylakoids in granae. It was shown that the thickness of granal thylakoids and the interspace between thylakoids increased comparing to control. Changes of the size and structure of thylakoids and granae in treated leaves may be associated with the enhanced accumulation of CO2 in the membrane. It is suggested that the carbonic anhydrase may also play a structural role in chloroplast granae.

  7. Synchrotron Radiation Provides a Plausible Explanation for the Generation of a Free Radical Adduct of Thioxolone in Mutant Carbonic Anhydrase II.

    Science.gov (United States)

    Sippel, Katherine H; Genis, Caroli; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Kiddle, James J; Tripp, Brian C; McKenna, Robert

    2010-10-07

    Thioxolone acts as a prodrug in the presence of carbonic anhydrase II (CA II), whereby the molecule is cleaved by thioester hydrolysis to the carbonic anhydrase inhibitor, 4-mercaptobenzene-1,3-diol (TH0). Thioxolone was soaked into the proton transfer mutant H64A of CA II in an effort to capture a reaction intermediate via X-ray crystallography. Structure determination of the 1.2 Å resolution data revealed the TH0 had been modified to a 4,4'-disulfanediyldibenzene-1,3-diol, a product of crystallization conditions, and a zinc ligated 2,4-dihydroxybenzenesulfenic acid, most likely induced by radiation damage. Neither ligand was likely a result of an enzymatic mechanism.

  8. Mono- and di-halogenated histamine, histidine and carnosine derivatives are potent carbonic anhydrase I, II, VII, XII and XIV activators.

    Science.gov (United States)

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

    2014-09-01

    Mono- and di-halogenated histamines, l-histidine methyl ester derivatives and carnosine derivatives incorporating chlorine, bromine and iodine were prepared and investigated as activators of five carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the transmembrane hCA XII and XIV. All of them were activated in a diverse manner by the investigated compounds, with a distinct activation profile.

  9. Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13.

    Science.gov (United States)

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

    2016-12-01

    A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co(2+) affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20 °C and pH of 8.3: kcat of 4.8 × 10(5) s(-1) and kcat/Km of 5.6 × 10(7) M(-1) × s(-1). This activity was potently inhibited by acetazolamide which showed a KI of 78.9 nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO3 biomineralization processes.

  10. 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.

  11. 1. alpha. ,25-dihydroxyvitamin D sub 3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells

    Energy Technology Data Exchange (ETDEWEB)

    Billecocq, A.; Emanuel, J.R.; Levenson, R.; Baron, R. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1990-08-01

    1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25(OH){sub 2}D{sub 3}), the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH){sub 2}D{sub 3} could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH){sub 2}D{sub 3} in a dose-and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH){sub 2}D{sub 3} was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH){sub 2}D{sub 3} regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.

  12. Cloning, characterization and anion inhibition study of a β-class carbonic anhydrase from the caries producing pathogen Streptococcus mutans.

    Science.gov (United States)

    Dedeoglu, Nurcan; De Luca, Viviana; Isik, Semra; Yildirim, Hatice; Kockar, Feray; Capasso, Clemente; Supuran, Claudiu T

    2015-07-01

    The oral pathogenic bacterium involved in human dental caries formation Streptococcus mutans, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the α- and the other one to the β-class. This last enzyme (SmuCA) has been cloned, characterized and investigated for its inhibition profile with a major class of CA inhibitors, the inorganic anions. Here we show that SmuCA has a good catalytic activity for the CO2 hydration reaction, with kcat 4.2×10(5)s(-1) and kcat/Km of 5.8×10(7)M(-1)×s(-1), being inhibited by cyanate, carbonate, stannate, divannadate and diethyldithiocarbamate in the submillimolar range (KIs of 0.30-0.64mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 15-46μM). The anion inhibition profile of the S. mutans enzyme is very different from other α- and β-CAs investigated earlier. Identification of effective inhibitors of this new enzyme may lead to pharmacological tools useful for understanding the role of S. mutans CAs in dental caries formation, and eventually the development of pharmacological agents with a new mechanism of antibacterial action.

  13. Expression of the CHOP-inducible carbonic anhydrase CAVI-b is required for BDNF-mediated protection from hypoxia.

    Science.gov (United States)

    Matthews, Tori A; Abel, Allyssa; Demme, Chris; Sherman, Teresa; Pan, Pei-wen; Halterman, Marc W; Parkkila, Seppo; Nehrke, Keith

    2014-01-16

    Carbonic anhydrases (CAs) comprise a family of zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide. CAs contribute to a myriad of physiological processes, including pH regulation, anion transport and water balance. To date, 16 known members of the mammalian alpha-CA family have been identified. Given that the catalytic family members share identical reaction chemistry, their physiologic roles are influenced greatly by their tissue and sub-cellular locations. CAVI is the lone secreted CA and exists in both saliva and the gastrointestinal mucosa. An alternative, stress-inducible isoform of CAVI (CAVI-b) has been shown to be expressed from a cryptic promoter that is activated by the CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP). The CAVI-b isoform is not secreted and is currently of unknown physiological function. Here we use neuronal models, including a model derived using Car6 and CHOP gene ablations, to delineate a role for CAVI-b in ischemic protection. Our results demonstrate that CAVI-b expression, which is increased through CHOP-signaling in response to unfolded protein stress, is also increased by oxygen-glucose deprivation (OGD). While enforced expression of CAVI-b is not sufficient to protect against ischemia, CHOP regulation of CAVI-b is necessary for adaptive changes mediated by BDNF that reduce subsequent ischemic damage. These results suggest that CAVI-b comprises a necessary component of a larger adaptive signaling pathway downstream of CHOP.

  14. 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.

  15. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the pathogenic yeast Candida glabrata with anions.

    Science.gov (United States)

    Innocenti, Alessio; Leewattanapasuk, Worraanong; Mühlschlegel, Fritz A; Mastrolorenzo, Antonio; Supuran, Claudiu T

    2009-08-15

    A beta-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomycescerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO(2) hydrase activity, with a k(cat) of 3.8 x 10(5)s(-1) and k(cat)/K(M) of 4.8 x 10(7)M(-1)s(-1). The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 0.60-1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K(I)s of 86-98 microM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K(I)s of 27-42 mM).

  16. Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase

    Energy Technology Data Exchange (ETDEWEB)

    J Mecinovic; P Snyder; K Mirica; S Bai; E Mack; R Kwant; D Moustakas; A Heroux; G Whitesides

    2011-12-31

    The hydrophobic effect, the free-energetically favorable association of nonpolar solutes in water, makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different but structurally similar hydrophobic groups, aliphatic hydrocarbons and aliphatic fluorocarbons, and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H{sub 2}NSO{sub 2}C{sub 6}H{sub 4}-CONHCH{sub 2}(CX{sub 2}){sub n}CX{sub 3}, n = 0-4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of nonoptimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and

  17. How does the exchange of one oxygen atom with sulfur affect the catalytic cycle of carbonic anhydrase?

    Science.gov (United States)

    Schenk, Stephan; Kesselmeier, Jürgen; Anders, Ernst

    2004-06-21

    We have extended our investigations of the carbonic anhydrase (CA) cycle with the model system [(H(3)N)(3)ZnOH](+) and CO(2) by studying further heterocumulenes and catalysts. We investigated the hydration of COS, an atmospheric trace gas. This reaction plays an important role in the global COS cycle since biological consumption, that is, uptake by higher plants, algae, lichens, and soil, represents the dominant terrestrial sink for this gas. In this context, CA has been identified by a member of our group as the key enzyme for the consumption of COS by conversion into CO(2) and H(2)S. We investigated the hydration mechanism of COS by using density functional theory to elucidate the details of the catalytic cycle. Calculations were first performed for the uncatalyzed gas phase reaction. The rate-determining step for direct reaction of COS with H(2)O has an energy barrier of deltaG=53.2 kcal mol(-1). We then employed the CA model system [(H(3)N)(3)ZnOH](+) (1) and studied the effect on the catalytic hydration mechanism of replacing an oxygen atom with sulfur. When COS enters the carbonic anhydrase cycle, the sulfur atom is incorporated into the catalyst to yield [(H(3)N)(3)ZnSH](+) (27) and CO(2). The activation energy of the nucleophilic attack on COS, which is the rate-determining step, is somewhat higher (20.1 kcal mol(-1) in the gas phase) than that previously reported for CO(2). The sulfur-containing model 27 is also capable of catalyzing the reaction of CO(2) to produce thiocarbonic acid. A larger barrier has to be overcome for the reaction of 27 with CO(2) compared to that for the reaction of 1 with CO(2). At a well-defined stage of this cycle, a different reaction path can emerge: a water molecule helps to regenerate the original catalyst 1 from 27, a process accompanied by the formation of thiocarbonic acid. We finally demonstrate that nature selected a surprisingly elegant and efficient group of reactants, the [L(3)ZnOH](+)/CO(2)/H(2)O system, that helps

  18. Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

    Science.gov (United States)

    Pedrozo, H. A.; Schwartz, Z.; Dean, D. D.; Harrison, J. L.; Campbell, J. W.; Wiederhold, M. L.; Boyan, B. D.

    1997-01-01

    To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

  19. Inhibition studies of bacterial, fungal and protozoan β-class carbonic anhydrases with Schiff bases incorporating sulfonamide moieties.

    Science.gov (United States)

    Ceruso, Mariangela; Carta, Fabrizio; Osman, Sameh M; Alothman, Zeid; Monti, Simona Maria; Supuran, Claudiu T

    2015-08-01

    A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three β-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high β-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.

  20. Inhibition of the alpha- and beta-carbonic anhydrases from the gastric pathogen Helycobacter pylori with anions.

    Science.gov (United States)

    Maresca, Alfonso; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2013-04-01

    The gastric pathogen Helicobacter pylori encodes two carbonic anhydrases (CAs, EC 4.2.1.1), an α- and a β-class one, hpαCA and hpβCA, crucial for its survival in the acidic environment from the stomach. Sulfonamides, strong inhibitors of these enzymes, block the growth of the pathogen, in vitro and in vivo. Here we report the inhibition of the two H. pylori CAs with inorganic and complex anions and other molecules interacting with zinc proteins. hpαCA was inhibited in the low micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid, and in the submillimolar one by cyanide, cyanate, hydrogen sulfide, divanadate, tellurate, perruthenate, selenocyanide, trithiocarbonate, iminodisulfonate. hpβCA generally showed a stronger inhibition with most of these anions, with several low micromolar and many submillimolar inhibitors detected. These inhibitors may be used as leads for developing anti-H. pylori agents with a diverse mechanism of action compared to clinically used antibiotics.

  1. 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.

  2. 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.

  3. The importance of carbonic anhydrase II in red blood cells during exposure of chicken embryos to CO2.

    Science.gov (United States)

    Everaert, N; Willemsen, H; Hulikova, A; Brown, H; Decuypere, E; Swietach, P; Bruggeman, V

    2010-07-31

    The importance of carbonic anhydrase (CA) during exposure of chicken embryos to CO(2) during the second half of incubation was investigated. The protein abundance and activity of CAII in erythrocytes was significantly higher in CO(2)-exposed embryos compared to normal conditions. Daily injections of acetazolamide (ATZ), an inhibitor of CA, increased blood P(CO2) and decreased blood pH in both control and CO(2)-incubated embryos. ATZ increased blood bicarbonate concentration in embryos exposed to normal atmosphere and in day-12 embryos exposed to high CO(2). The tendency of an increased blood potassium concentration in ATZ-injected embryos under standard atmospheric conditions might indicate that protons were exchanged with intracellular potassium. However, there was no evidence for such an exchange in CO(2)-incubated ATZ-treated embryos. This study shows for the first time that chicken embryos adapt to CO(2) during the second half of incubation by increasing CAII protein expression and function in red blood cells. This response may serve to "buffer" elevated CO(2) levels.

  4. Poly(amidoamine) dendrimers show carbonic anhydrase inhibitory activity against α-, β-, γ- and η-class enzymes.

    Science.gov (United States)

    Carta, Fabrizio; Osman, Sameh M; Vullo, Daniela; AlOthman, Zeid; Del Prete, Sonia; Capasso, Clemente; Supuran, Claudiu T

    2015-11-01

    Four generations of poly(amidoamine) (PAMAM) dendrimers incorporating benzenesulfonamide moieties were investigated as inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β-, γ- and η-classes which are present in pathogenic bacteria, fungi or protozoa. The following bacterial, fungal and protozoan organisms were included in the study: Vibrio cholerae, Trypanosoma cruzi, Leishmania donovani chagasi, Porphyromonas gingivalis, Cryptococcus neoformans, Candida glabrata, and Plasmodium falciparum. The eight pathozymes present in these organisms were efficiently inhibited by the four generations PAMAM-sulfonamide dendrimers, but multivalency effects were highly variable among the different enzyme classes. The Vibrio enzyme VchCA was best inhibited by the G3 dendrimer incorporating 32 sulfamoyl moieties. The Trypanosoma enzyme TcCA on the other hand was best inhibited by the first generation dendrimer G0 (with 4 sulfamoyl groups), whereas for other enzymes the optimal inhibitory power was observed for the G1 or G2 dendrimers, with 8 and 16 sulfonamide functionalities. This study thus proves that the multivalency may be highly relevant for enzyme inhibition for some but not all CAs from pathogenic organisms. On the other hand, some dendrimers investigated here showed a better inhibitory power compared to acetazolamide for enzymes from widespread pathogens, such as the η-CA from Plasmodium falciparum. Overall, the main conclusion is that this class of molecules may lead to important developments in the field of anti-infective CA inhibitors.

  5. A new class of quinazoline-sulfonamides acting as efficient inhibitors against the α-carbonic anhydrase from Trypanosoma cruzi.

    Science.gov (United States)

    Alafeefy, Ahmed M; Ceruso, Mariangela; Al-Jaber, Nabila A; Parkkila, Seppo; Vermelho, Alane Beatriz; Supuran, Claudiu T

    2015-01-01

    The protozoan parasite Trypanosoma cruzi is the agent responsible for trypanosomiasis (Chagas disease) in humans and other animals. It has been recently reported that this pathogen encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), denominated TcCA, which was shown to be crucial for its life cycle. Inhibition studies of a class of 4-oxoquinazoline containing a benzensulfonamide moiety and their 4-thioxo bioisosteres against the protozoan enzyme TcCA are described here. Most of 4-oxoquinazoline sulfonamides showed nanomolar TcCA inhibition activity with K(I)s in the same order of magnitude of acetazolamide (AAZ), whereas their thioxo bioisosters showed moderate anti-Trypanosoma CA potency with K(I)s in the micromolar range. The discovery of compounds incorporating a 4-oxoquinazoline ring as a low-nanomolar TcCA inhibitor is quite promising and it may be useful for developing anti-Trypanosoma agents with a novel mechanism of action compared to the clinically used drugs (such as benznidazole, nifurtimox) for which significant resistance and serious adverse effects due to their high-toxicity appeared.

  6. 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.

  7. Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

    Science.gov (United States)

    Osborn, Hannah L; Alonso-Cantabrana, Hugo; Sharwood, Robert E; Covshoff, Sarah; Evans, John R; Furbank, Robert T; von Caemmerer, Susanne

    2017-01-01

    In C4 species, the major β-carbonic anhydrase (β-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3(-), which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in β-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C(18)O(16)O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

  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. Cloning, characterization and sulfonamide inhibition studies of an α-carbonic anhydrase from the living fossil sponge Astrosclera willeyana.

    Science.gov (United States)

    Ohradanova, Anna; Vullo, Daniela; Pastorekova, Silvia; Pastorek, Jaromir; Jackson, Daniel J; Wörheide, Gert; Supuran, Claudiu T

    2012-02-15

    The α-carbonic anhydrase (CA, EC 4.2.1.1) Astrosclerin-3 previously isolated from the living fossil sponge Astrosclera willeyana (Jackson et al., Science 2007, 316, 1893), was cloned, kinetically characterized and investigated for its inhibition properties with sulfonamides and sulfamates. Astrosclerin-3 has a high catalytic activity for the CO(2) hydration reaction to bicarbonate and protons (k(cat) of 9.0×10(5) s(-1) and k(cat)/K(m) of 1.1×10(8) M(-1) × s(-1)), and is inhibited by various aromatic/heterocyclic sulfonamides and sulfamates with inhibition constants in the range of 2.9 nM-8.85 μM. Astrosclerin, and the human isoform CA II, display similar kinetic properties and affinities for sulfonamide inhibitors, despite more than 550 million years of independent evolution. Because Astrosclerin-3 is involved in biocalcification, the inhibitors characterized here may be used to gain insights into such processes in other metazoans.

  10. Characterization and anions inhibition studies of an α-carbonic anhydrase from the teleost fish Dicentrarchus labrax.

    Science.gov (United States)

    Ekinci, Deniz; Ceyhun, Saltuk Buğrahan; Sentürk, Murat; Erdem, Deryanur; Küfrevioğlu, Omer İrfan; Supuran, Claudiu T

    2011-01-15

    Carbonic anhydrase (CA; EC 4.2.1.1) was purified from the gill of the teleost fish Dicentrarchus labrax (European seabass). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 84.9-fold with a yield of 58%, and a specific activity of 838.9 U/mg proteins. It has an optimum pH at 8.0; an optimum temperature at 10°C. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate. The following anions, H₂NSO₃⁻, I⁻, SCN⁻, NO₃⁻, NO₂⁻, N₃⁻, Br⁻, Cl⁻, SO₄²⁻, and F⁻ showed inhibitory effects on the enzyme. Sulfamic acid, iodide, and thiocyanate exhibited the strongest inhibitory action, in the micromolar range (K(i)s of 87-187 μM). NO₃⁻, NO₂⁻ and N₃⁻ were moderate inhibitors, whereas other anions showed only weak actions. All tested anions inhibited the enzyme in a competitive manner. Our findings indicate that these anions inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various anions differs significantly between the fish and mammalian CAs.

  11. 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.

  12. Carbonic anhydrase activity in the vas deferens of the cotton leafworm - Spodoptera littoralis (Lepidoptera: Noctuidae) controlled by circadian clock.

    Science.gov (United States)

    Kotwica, J; Ciuk, M A; Joachimiak, E; Rowinski, S; Cymborowski, B; Bebas, P

    2006-11-01

    The male reproductive tract of Lepidoptera is an ideal model for the study of the physiological role of peripheral clocks in insects. The latter are significant in the generation and coordination of rhythmic phenomena which facilitate the initial stages of sperm capacitation. This process requires the maintenance of pH in the upper vas deferens (UVD) aided by, among others, H+-ATPase. Our aim was to determine the potential involvement of carbonic anhydrase (CA) in this process, an enzyme tasked with generating protons subsequently utilized by H+-ATPase to acidify the UVD milieu in S. littoralis, during the time when the lumen of this organ is filled with sperm. We attempted to answer the question whether CA activity can be controlled by the biological oscillator present in the male reproductive tract of the cotton leafworm. Using PAGE zymography, the presence of CA was demonstrated in the UVD wall, but not in the luminal fluid nor in the sperm. Using histochemistry, it was shown that CA is active in the UVD epithelium, and that this activity varies throughout the day and is most likely controlled by an endogenous biological clock. Conversely, the application of CA inhibitors, acetazolamide and sodium thiocyanate, in conjunction with an analysis of H+-ATPase activity in the acidification the UVD environment shows that CA most likely does not play a direct role in the regulation of the pH in this organ.

  13. 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.

  14. Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

    Science.gov (United States)

    Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

    2017-01-20

    The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol(-1) , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pKa values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work.

  15. Degradation of carbonyl sulfide by Actinomycetes and detection of clade D of β-class carbonic anhydrase.

    Science.gov (United States)

    Ogawa, Takahiro; Kato, Hiromi; Higashide, Mitsuru; Nishimiya, Mami; Katayama, Yoko

    2016-09-25

    Carbonyl sulfide (COS) is an atmospheric trace gas and one of the sources of stratospheric aerosol contributing to climate change. Although one of the major sinks of COS is soil, the distribution of COS degradation ability among bacteria remains unclear. Seventeen out of 20 named bacteria belonging to Actinomycetales had COS degradation activity at mole fractions of 30 parts per million by volume (ppmv) COS. Dietzia maris NBRC 15801(T) and Mycobacterium sp. THI405 had the activity comparable to a chemolithoautotroph Thiobacillus thioparus THI115 that degrade COS by COS hydrolase for energy production. Among 12 bacteria manifesting rapid degradation at 30 ppmv COS, Dietzia maris NBRC 15801(T) and Streptomyces ambofaciens NBRC 12836(T) degraded ambient COS (∼500 parts per trillion by volume). Geodermatophilus obscurus NBRC 13315(T) and Amycolatopsis orientalis NBRC 12806(T) increased COS concentrations. Moreover, six of eight COS degrading bacteria isolated from soils had partial nucleotide sequences similar to that of the gene encoding clade D of β-class carbonic anhydrase, which included COS hydrolase. These results indicate the potential importance of Actinomycetes in the role of soils as sinks of atmospheric COS.

  16. Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

    Science.gov (United States)

    Perfetto, Rosa; Del Prete, Sonia; Vullo, Daniela; Sansone, Giovanni; Barone, Carmela; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2017-12-01

    A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 10(5) s(-1) and kcat/Km of 3.6 × 10(7) M(-1) × s(-1). The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

  17. Biomimetic CO2 capture using a highly thermostable bacterial α-carbonic anhydrase immobilized on a polyurethane foam.

    Science.gov (United States)

    Migliardini, Fortunato; De Luca, Viviana; Carginale, Vincenzo; Rossi, Mosè; Corbo, Pasquale; Supuran, Claudiu T; Capasso, Clemente

    2014-02-01

    The biomimetic approach represents an interesting strategy for carbon dioxide (CO2) capture, offering advantages over other methods, due to its specificity for CO2 and its eco-compatibility, as it allows concentration of CO2 from other gases, and its conversion to water soluble ions. This approach uses microorganisms capable of fixing CO2 through metabolic pathways or via the use of an enzyme, such as carbonic anhydrase (CA, EC 4.2.1.1). Recently, our group cloned and purified a novel bacterial α-CA, named SspCA, from the thermophilic bacteria, Sulfurihydrogenibium yellowstonense YO3AOP1 living in hot springs at temperatures of up to 110 °C. This enzyme showed an exceptional thermal stability, retaining its high catalytic activity for the CO2 hydration reaction even after being heated at 70 °C for several hours. In the present paper, the SspCA was immobilized within a polyurethane (PU) foam. The immobilized enzyme was found to be catalytically active and showed a long-term stability. A bioreactor containing the "PU-immobilized enzyme" (PU-SspCA) as shredded foam was used for experimental tests aimed to verify the CO2 capture capability in conditions close to those of a power plant application. In this bioreactor, a gas phase, containing CO2, was put into contact with a liquid phase under conditions, where CO2 contained in the gas phase was absorbed and efficiently converted into bicarbonate by the extremo-α-CA.

  18. Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria.

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    Hongo, Yuki; Nakamura, Yoshimitsu; Shimamura, Shigeru; Takaki, Yoshihiro; Uematsu, Katsuyuki; Toyofuku, Takashi; Hirayama, Hisako; Takai, Ken; Nakazawa, Masatoshi; Maruyama, Tadashi; Yoshida, Takao

    2013-12-01

    Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + H(+), and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE and LC-MS/MS. We found that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii, which showed that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a non-symbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.

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

    Science.gov (United States)

    Sedlakova, Olga; Svastova, Eliska; Takacova, Martina; Kopacek, Juraj; Pastorek, Jaromir; Pastorekova, Silvia

    2014-01-08

    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 and/or acidosis.

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

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    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

  1. Carbonic anhydrase 2-like and Na⁺-K⁺-ATPase α gene expression in medaka (Oryzias latipes) under carbonate alkalinity stress.

    Science.gov (United States)

    Yao, Zongli; Lai, Qifang; Hao, Zhuoran; Chen, Ling; Lin, Tingting; Zhou, Kai; Wang, Hui

    2015-12-01

    High carbonate alkalinity is one of the major stress factors for living organisms in saline-alkaline water areas. Acute and chronic effects of carbonate alkalinity on expression of two genes, carbonic anhydrase 2-like (CA2-like) and Na(+)-K(+)-ATPase α subunit (NKA-α) mRNA in medaka (Oryzias latipes) were evaluated to better understand the responses important for coping with a carbonate alkalinity stress. In the acute exposure experiment, the expression of CA2-like and NKA-α mRNA in the gill and kidney of medaka were examined from 0 h to 7 days exposed to 30.4 mM carbonate alkalinity water. Exposure to high carbonate alkalinity resulted in a transitory alkalosis, followed by a transient increase in gill and kidney CA2-like and NKA-α mRNA expression. In the chronic exposure experiment, the expression of these two genes was examined in the gill and kidney at 50 days post-exposure to six different carbonate alkalinity concentrations ranging from 1.5 to 30.4 mM. Gill and kidney CA2-like mRNA levels in 30.4 mM were approximately 10 and 30 times higher than that of the control (1.5 mM), respectively. Less differences were found in NKA-α expression in the 50-days exposure. The results indicate that when transferred to high carbonate alkalinity water, a transitory alkalosis may occur in medaka, followed by compensatory acid-base and ion regulatory responses. Thus, CA2-like and NKA-α are at least two of the important factors that contribute to the regulation of alkalinity stress.

  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.

  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. 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.

  5. Relationship among salivary carbonic anhydrase VI activity and flow rate, biofilm pH and caries in primary dentition.

    Science.gov (United States)

    Frasseto, F; Parisotto, T M; Peres, R C R; Marques, M R; Line, S R P; Nobre Dos Santos, M

    2012-01-01

    This study aimed to determine the activity of carbonic anhydrase isoenzyme VI (CAVI) in the saliva of preschool children with caries and to investigate the relationship between caries and salivary CAVI activity, salivary flow rate and biofilm pH before and after a 20% sucrose rinse. Thirty preschool children aged 45.3-80.3 months were divided into two groups: a caries-free group and a caries group. Clinical examinations were conducted by one examiner (κ = 0.95) according to WHO criteria (dmfs) and early caries lesions. From each subject, CAVI activity, salivary flow rate and plaque pH were determined before and after a sucrose rinse. The results were submitted to Wilcoxon, Mann-Whitney and Spearman correlation tests (α = 0.05). The results showed that prerinse CAVI activity and its variation were higher in the saliva from caries children than from caries-free children. No difference was found between the two groups in postrinse salivary CAVI activity. After rinsing, biofilm pH differences were lower in both groups (p = 0.0012 and p = 0.0037 for the caries and caries-free groups, respectively). Also, after the sucrose rinse, salivary flow rate significantly increased in caries and caries-free groups (p = 0.0003, p = 0.0037). The variation of salivary CAVI activity was negatively correlated with caries (r = -0.501, p = 0.005). Child's age showed a positive correlation with caries (r = 0.456, p = 0.011). These results suggest that variation of salivary CAVI activity and child's age are associated with dental caries in preschool children.

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

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    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.

  7. Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation▿

    Science.gov (United States)

    Kalloniati, Chrysanthi; Tsikou, Daniela; Lampiri, Vasiliki; Fotelli, Mariangela N.; Rennenberg, Heinz; Chatzipavlidis, Iordanis; Fasseas, Costas; Katinakis, Panagiotis; Flemetakis, Emmanouil

    2009-01-01

    Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3− produced by CAA1. PMID:19218391

  8. Characterization of a Mesorhizobium loti alpha-type carbonic anhydrase and its role in symbiotic nitrogen fixation.

    Science.gov (United States)

    Kalloniati, Chrysanthi; Tsikou, Daniela; Lampiri, Vasiliki; Fotelli, Mariangela N; Rennenberg, Heinz; Chatzipavlidis, Iordanis; Fasseas, Costas; Katinakis, Panagiotis; Flemetakis, Emmanouil

    2009-04-01

    Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.

  9. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

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    Janine Hallerdei

    Full Text Available We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  10. Evidence for an early evolutionary emergence of γ-type carbonic anhydrases as components of mitochondrial respiratory complex I

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    Gray Michael W

    2010-06-01

    Full Text Available Abstract Background The complexity of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to γ-type carbonic anhydrases (γCA have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution. Results We have performed a tandem mass spectrometric characterization of CI from the amoeboid protozoon Acanthamoeba castellanii. Among the proteins identified were two γCA homologs, AcCa1 and AcCa2, demonstrating that γCA proteins are not specific to plants/green algae. In fact, through bioinformatics searches we detected γCA homologs in diverse protist lineages, and several of these homologs are predicted to possess N-terminal mitochondrial targeting peptides. Conclusions The detection of γCAs in CI of Acanthamoeba, considered to be a closer relative of animals and fungi than plants, suggests that γCA proteins may have been an ancestral feature of mitochondrial CI, rather than a novel, plant-specific addition. This assertion is supported by the presence of genes encoding γCAs in the nuclear genomes of a wide variety of eukaryotes. Together, these findings emphasize the importance of a phylogenetically broad characterization of CI for elucidating CI evolution in eukaryotes.

  11. Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism.

    Science.gov (United States)

    Shah, Gul N; Rubbelke, Timothy S; Hendin, Joshua; Nguyen, Hien; Waheed, Abdul; Shoemaker, James D; Sly, William S

    2013-04-30

    Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium-potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A/B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-than-predicted numbers even when supplemented with sodium-potassium citrate in their drinking water. Survival after weaning was also reduced, especially for males. In addition, fasting blood glucose levels for DKO mice were significantly lower than for controls (153 ± 33 vs. 230 ± 24 mg/dL). The enhanced hyperammonemia and lower fasting blood sugar, which are both seen in the DKO mice, indicate that both Car5A and Car5B contribute to both ammonia detoxification (ureagenesis) and regulation of fasting blood sugar (gluconeogenesis). Car5A, which is expressed mainly in liver, clearly has the predominant role in ammonia detoxification. The contribution of Car5B to ureagenesis and gluconeogenesis was evident only on a Car5A null background.

  12. C4 photosynthesis evolution in the transitional grass Neurachne: loss of a carbonic anhydrase chloroplast transit peptide.

    Science.gov (United States)

    Clayton, Harmony; Saladié, Montserrat; Rolland, Vivien; Sharwood, Robert E; Macfarlane, Terry; Ludwig, Martha

    2017-02-02

    Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the cDNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), N. minor (C3-C4), and N. alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a, CA1b, CA2a and CA2b. Transcript analyses found CA1 mRNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, comprising approximately 99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11 amino acid deletion in the N-terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting chloroplast targeting of CA1a is the ancestral state, and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne. Remarkably, this mechanism is homoplastic with evolution of the C4-associated CA in the dicotyledonous Flaveria, although the actual mutations in the two lineages differ.

  13. Mitochondrial carbonic anhydrase CA VB: Differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles

    OpenAIRE

    Shah, Gul N.; Hewett-Emmett, David; Grubb, Jeffrey H.; Migas, Mary C.; Fleming, Robert E.; Waheed, Abdul; Sly, William S.

    2000-01-01

    A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitoc...

  14. 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

  15. 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.

  16. 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.

  17. A High-Resolution Crystal Structure of a Psychrohalophilic α–Carbonic Anhydrase from Photobacterium profundum Reveals a Unique Dimer Interface

    Energy Technology Data Exchange (ETDEWEB)

    Somalinga, Vijayakumar; Buhrman, Greg; Arun, Ashikha; Rose, Robert B.; Grunden, Amy M. (NCSU)

    2016-12-09

    Bacterial α–carbonic anhydrases (α-CA) are zinc containing metalloenzymes that catalyze the rapid interconversion of CO2 to bicarbonate and a proton. We report the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, Photobacterium profundum. Size exclusion chromatography of the purified P. profundum α–CA (PprCA) reveals that the protein is a heterogeneous mix of monomers and dimers. Furthermore, an “in-gel” carbonic anhydrase activity assay, also known as protonography, revealed two distinct bands corresponding to monomeric and dimeric forms of PprCA that are catalytically active. The crystal structure of PprCA was determined in its native form and reveals a highly conserved “knot-topology” that is characteristic of α–CA’s. Similar to other bacterial α–CA’s, PprCA also crystallized as a dimer. Furthermore, dimer interface analysis revealed the presence of a chloride ion (Cl-) in the interface which is unique to PprCA and has not been observed in any other α–CA’s characterized so far. Molecular dynamics simulation and chloride ion occupancy analysis shows 100% occupancy for the Cl- ion in the dimer interface. Zinc coordinating triple histidine residues, substrate binding hydrophobic patch residues, and the hydrophilic proton wire residues are highly conserved in PprCA and are identical to other well-studied α–CA’s.

  18. Functional role of a putative carbonic anhydrase II-binding domain in the electrogenic Na+ -HCO₃- cotransporter NBCe1 expressed in Xenopus oocytes.

    Science.gov (United States)

    Yamada, Hideomi; Horita, Shoko; Suzuki, Masashi; Fujita, Toshiro; Seki, George

    2011-01-01

    The electrogenic Na+ -HCO₃⁻ cotransporter NBCe1 plays essential roles in the regulation of systemic and/or local pH. Homozygous inactivating mutations in NBCe1 cause proximal renal tubular acidosis associated with ocular abnormalities. We recently showed that defective membrane expression of NBCe1, caused by several mutations such as Delta65bp (S982NfsX4), is also associated with familial migraine. The Delta65bp mutant is quite unique in that it lacks a putative carbonic anhydrase (CA) II-binding domain but still shows an apparently normal transport activity in Xenopus oocytes. In this addendum, we show that the co-expression of CAII together with the wild-type NBCe1 or the Delta65bp mutant does not enhance the NBCe1 activities in oocytes. Moreover, a carbonic anhydrase inhibitor acetazolamide fails to inhibit the wild-type or the Delta65bp activities co-expressed with CAII. These results indicate that a bicarbonate transport metabolon proposed for the interaction between CAII and NBCe1 does not work at least in Xenopus oocytes.

  19. 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.

  20. Synthesis, characterization and in vitro inhibition of metal complexes of pyrazole based sulfonamide on human erythrocyte carbonic anhydrase isozymes I and II.

    Science.gov (United States)

    Büyükkıdan, Nurgün; Büyükkıdan, Bülent; Bülbül, Metin; Kasımoğulları, Rahmi; Mert, Samet

    2017-12-01

    Sulfonamides represent an important class of biologically active compounds. A sulfonamide possessing carbonic anhydrase (CA) inhibitory properties obtained from a pyrazole based sulfonamide, ethyl 1-(3-nitrophenyl)-5-phenyl-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)-1H-pyrazole-4-carboxylate (1), and its metal complexes with the Ni(II) for (2), Cu(II) for (3) and Zn(II) for (4) have been synthesized. The structures of metal complexes (2-4) were established on the basis of their elemental analysis, (1)H NMR, IR, UV-Vis and MS spectral data. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes I and II, with 1 and synthesized complexes (2-4) and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. The complexes 2, 3 and 4 showed inhibition constant in the range 0.1460-0.3930 µM for hCA-I and 0.0740-0.0980 µM for hCA-II, and they had effective more inhibitory activity on hCA-I and hCA-II than corresponding free ligand 1 and than AAZ.

  1. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

    Science.gov (United States)

    In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

  2. 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.

  3. 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

  4. Research and application progress of carbonic anhydrase-immobilized technology%碳酸酐酶固定化技术研究与应用进展

    Institute of Scientific and Technical Information of China (English)

    潘富军; 周作明

    2014-01-01

    Currently,the main drawbacks of the most available amine-based processes for post-combustion CO2 are amine degradation and parasitic power loss.A novel Integrated Vacuum Carbonate Absorption Process (IVCAP)was proposed to reduce the energy usage by employing a potassium carbonate aqueous solution as a solvent for CO2 absorption.However,K2 CO3 -based system has a much slower CO2 absorption rate than amine-based system does.Carbonic anhydrase is the most effective known enzyme by far that catalyzes the hydration of CO2 into bicarbonate and a proton.The research and applications of immobilized carbonic anhydrase were reviewed,and the application prospect of IVCAP process improvement for carbon dioxide capture by selecting magnetic carrier material was presented.%目前以有机胺为吸收剂脱除 CO2的化学吸收法多存在氨基易分解、再生能耗高等不足。新型的 IVCAP 工艺采用碳酸钾水溶液吸收 CO2,可大幅节能降耗,但吸收速率较有机胺慢。碳酸酐酶是至今发现的最有效的 CO2水合酶催化剂,将该酶添加至 IVCAP 工艺中,可显著提高 CO2吸收速率。文中综述了固定化碳酸酐酶在碳捕集与封存技术中的研究和应用进展,并对其选用磁性载体材料强化 IVCAP 工艺对 CO2的捕集应用前景进行了展望。

  5. Engineering of carbon catabolite repression in recombinant xylose fermenting Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Roca, Christophe Francois Aime; Haack, Martin Brian; Olsson, Lisbeth

    2004-01-01

    Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (Deltamig1, XYL1, XYL2, XKS1) and CPB.MBH2 (Deltamig1, Deltamig2, XYL1, XYL2, XKS1) were...... of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting...

  6. Feedforward non-Michaelis-Menten mechanism for CO(2) uptake by Rubisco: contribution of carbonic anhydrases and photorespiration to optimization of photosynthetic carbon assimilation.

    Science.gov (United States)

    Igamberdiev, Abir U; Roussel, Marc R

    2012-03-01

    Rubisco, the most abundant protein serving as the primary engine generating organic biomass on Earth, is characterized by a low catalytic constant (in higher plants approx. 3s(-1)) and low specificity for CO(2) leading to photorespiration. We analyze here why this enzyme evolved as the main carbon fixation engine. The high concentration of Rubisco exceeding the concentration of its substrate CO(2) by 2-3 orders of magnitude makes application of Michaelis-Menten kinetics invalid and requires alternative kinetic approaches to describe photosynthetic CO(2) assimilation. Efficient operation of Rubisco is supported by a strong flux of CO(2) to the chloroplast stroma provided by fast equilibration of bicarbonate and CO(2) and forwarding the latter to Rubisco reaction centers. The main part of this feedforward mechanism is a thylakoidal carbonic anhydrase associated with photosystem II and pumping CO(2) from the thylakoid lumen in coordination with the rate of electron transport, water splitting and proton gradient across the thylakoid membrane. This steady flux of CO(2) limits photosynthesis at saturating CO(2) concentrations. At low ambient CO(2) and correspondingly limited capacity of the bicarbonate pool in the stroma, its depletion at the sites of Rubisco is relieved by utilizing O(2) instead of CO(2), i.e. by photorespiration, a process which supplies CO(2) back to Rubisco and buffers the redox state and energy level in the chloroplast. Thus, the regulation of Rubisco function aims to keep steady non-equilibrium levels of CO(2), NADPH/NADP and ATP/ADP in the chloroplast stroma and to optimize the condition of homeostatic photosynthetic flux of matter and energy.

  7. 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.

  8. Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

    OpenAIRE

    Larsson, C; von Stockar, U.; Marison, I; Gustafsson, L.

    1993-01-01

    Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a c...

  9. Design and Synthesis of Novel Nonsteroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis.

    Science.gov (United States)

    Bua, Silvia; Di Cesare Mannelli, Lorenzo; Vullo, Daniela; Ghelardini, Carla; Bartolucci, Gianluca; Scozzafava, Andrea; Supuran, Claudiu T; Carta, Fabrizio

    2017-02-09

    We report the synthesis of a series of hybrid compounds incorporating 6- and 7-substituted coumarins (carbonic anhydrase, CA inhibitors) derivatized with clinically used NSAIDs (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA overexpressed hCA IX and XII, with KI values in the low nanomolar-subnanomolar ranges. The antihyperalgesic activity of such compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model. Among all tested compounds, the 7-coumarine hybrid with ibuprofen showed potent and persistent antihyperalgesic effect up to 60 min after administration.

  10. 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.

  11. Synthesis and in Vivo Biological Evaluation of (68)Ga-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography.

    Science.gov (United States)

    Sneddon, Deborah; Niemans, Raymon; Bauwens, Matthias; Yaromina, Ala; van Kuijk, Simon J A; Lieuwes, Natasja G; Biemans, Rianne; Pooters, Ivo; Pellegrini, Paul A; Lengkeek, Nigel A; Greguric, Ivan; Tonissen, Kathryn F; Supuran, Claudiu T; Lambin, Philippe; Dubois, Ludwig; Poulsen, Sally-Ann

    2016-07-14

    Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

  12. Effect of high concentration of inert cosolutes on the refolding of an enzyme: carbonic anhydrase B in sucrose and ficoll 70.

    Science.gov (United States)

    Monterroso, Begoña; Minton, Allen P

    2007-11-16

    The kinetics of refolding of carbonic anhydrase II following transfer from a buffer containing 5 m guanidinium chloride to a buffer containing 0.5 m guanidinium chloride were studied by measuring the time-dependent recovery of enzymatic activity. Experiments were carried out in buffer containing concentrations of two "inert" cosolutes, sucrose and Ficoll 70, a sucrose polymer, at concentrations up to 150 g/liter. Data analysis indicates that both cosolutes significantly accelerate the rate of refolding to native or compact near-native conformations, but decrease the fraction of catalytically active enzyme recovered in the limit of long time. According to the simplest model that fits the data, both cosolutes accelerate a competing side reaction yielding inactive compact species. Acceleration of the side reaction by Ficoll is significantly greater than that of sucrose at equal w/v concentrations.

  13. 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.

  14. 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.

  15. Physiological function, diversity of carbonic anhydrase and its application%碳酸酐酶的生理功能、多样性及其在CO2捕集中的应用

    Institute of Scientific and Technical Information of China (English)

    李春秀; 姜笑辰; 邱勇隽; 许建和

    2013-01-01

    碳酸酐酶(carbonic anhydrase)作为一种活性中心含有锌离子的金属酶,能够可逆催化CO2生成碳酸氢盐的水合反应,该反应在生物体内承担着多样的生理学功能,具有高度的生物学意义.除广泛存在于真核生物以外,该酶在淡水、海水、嗜常温、嗜热、厌氧、好氧、致病、产酸、自养、异养等多种原核微生物中也有广泛的分布,并参与光合作用、呼吸作用和以CO2作为底物的反应,维持生理pH以及离子转运等生理过程.近年来,随着温室效应的日益加剧,生物固定CO2作为该酶的一种全新应用引起了研究者的广泛关注.回顾了碳酸酐酶作为催化剂参与CO2固定过程的历史、现状和最新发现,同时展望了未来应用的趋势.%As a metalloenzyme combining a Zn2+ at its active centers,carbonic anhydrase catalyze reaction between CO2 and H2O reversibly, the reaction can undertakes various physiological functions in organisms. Carbonic anhydrase can exist in the eukaryote, and in diversity of prokaryotes. Evidence for the presence of carbonic anhydrase is obtained for freshwater, marine, mesophilic, thermophilic, aerobic, anaerobic, pathogenic, symbiotic, acetogenic, autotrophic, heterotrophic, and photosynthetic species. In prokaryotes, carbonic anhydrases are involved in various biochemical and physiological processes, including photosynthesis, respiration, ion transport, and CO2 and bicarbonate balance. While anthropogenic CO2 emission has led to adverse impact on climate and has been implicated in global warming, this enzyme has found a new dimension in the field of biomimetic CO2 sequestration. This paper reviews the research advances on carbonic anhydrase-driven processes for CO2 sequestration research and engineering and, suggests the possible future directions in application.

  16. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

    Science.gov (United States)

    Al-Haideri, Halah; White, Michael A; Kelly, David J

    2016-02-01

    Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni.

  17. The enzyme-inhibitor approach to cell-selective labelling. Pt. 1; Sulphonamide inhibitors of carbonic anhydrase as carriers for red cell labelling: in vitro uptake of pIBS by human red blood cells

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    Singh, Jaspal; Wyeth, P. (Southampton Univ. (UK))

    1991-01-01

    Red cell carbonic anhydrase is identified as an ideal target in an enzyme-inhibitor approach to radiolabel localisation. Current problems in blood pool labelling could be overcome by using selective sulphonamide inhibitors as carriers. p-Iodobenzenesulphonamide (pIBS) was selected as the choice reagent for red blood cell labelling. Rapid uptake of ({sup 125}I)-pIBS was found in vitro, consistent with passive diffusion across the cell membrane. The intracellular binding could be attributed to interaction with two specific acceptor sites, with dissociation constants of 4.9 +- 1.0 and 0.10+- 0.05 {mu}mol dm{sup -3}, and maximum binding capacities of 166 +- 5 and 19.9 +- 1.0 {mu}mol dm{sup -3}, respectively under the experimental conditions. These data correlate with the two major carbonic anhydrase isozymes; acceptor assignments were confirmed by gel chromatography of the red cell lysate. (author).

  18. Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.

    Science.gov (United States)

    Van Heeke, G; Shaw, R; Schnizer, R; Couton, J M; Schuster, S M; Wagner, F W

    1993-08-01

    A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.

  19. The cellular response of Saccharomyces cerevisiae to multi-walled carbon nanotubes (MWCNTs

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    Chantelle L. Phillips

    2015-03-01

    Full Text Available Nanoparticles (NPs especially those of carbon nanotubes (CNTs have remarkable properties that are very desirable in various biological and biomedical applications. This has necessitated the rapid study of CNT toxicities, to augment their safe use, particularly, in yeast cells. The yeast cell; Saccharomyces cerevisiae is a widely used industrial and biological organism with very limited data regarding their cellular behaviour in NPs. The current study examines the cellular response of S. cerevisiae to MWCNTs. The CNTs were produced by the swirled floating catalytic chemical vapour deposition (SFCCVD method and covalently functionalised using 1,3-dipolar cycloaddition. The CNT properties such as size, surface area, quality and surface vibrations were characterized using TEM, SEM, BET, TGA and Raman spectroscopy, respectively. The cellular uptake was confirmed with a FITC functionalised MWCNTs using 1H NMR, SEM and TEM. The CNT concentrations of 2–40 μg/ml were used to determine the cellular response through cell growth phases and cell viability characteristics. The TEM and SEM analyses showed the production of MWCNTs with an average diameter of 53 ± 12 nm and a length of 2.5 ± 0.5 μm. The cellular uptake of FITC-MWCNTs showed 100% internalisation in the yeast cells. The growth curve responses to the MWCNT doses showed no significant differences at P > 0.05 on the growth rate and viability of the S. cerevisiae cells.

  20. Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

    Science.gov (United States)

    Pinu, Farhana R; Edwards, Patrick J B; Gardner, Richard C; Villas-Boas, Silas G

    2014-12-01

    To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine.

  1. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with novel Schiff bases: identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones.

    Science.gov (United States)

    Sarikaya, Busra; Ceruso, Mariangela; Carta, Fabrizio; Supuran, Claudiu T

    2014-11-01

    A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.

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

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    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.

  3. 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.

  4. Design and synthesis of benzothiazole-6-sulfonamides acting as highly potent inhibitors of carbonic anhydrase isoforms I, II, IX and XII.

    Science.gov (United States)

    Ibrahim, Diaa A; Lasheen, Deena S; Zaky, Maysoun Y; Ibrahim, Amany W; Vullo, Daniela; Ceruso, Mariangela; Supuran, Claudiu T; Abou El Ella, Dalal A

    2015-08-01

    A series of novel 2-aminobenzothiazole derivatives bearing sulfonamide at position 6 was designed, synthesized and investigated as inhibitors of four isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), the cytosolic CA I and II, and the tumor-associated isozymes CA IX and XII. Docking and binding energy studies were carried out to reveal details regarding the favorable interactions between the scaffolds of these new inhibitors and the active sites of the investigated CA isoforms. Most of the novel compounds were acting as highly potent inhibitors of the tumor-associated hCA IX and hCA XII with KIs in the nanomolar range. The ubiquitous and dominant rapid cytosolic isozyme hCA II was also inhibited with KIs ranging from 3.5 to 45.4 nM. The favorable interactions between some of the new compounds and the active site of different CA isoforms were delineated by using molecular docking which may be useful for designing compounds with high affinity and selectivity for some CAs with biomedical applications.

  5. 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.

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    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.

  6. 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.

  7. Cloning, characterization, and sulfonamide and thiol inhibition studies of an α-carbonic anhydrase from Trypanosoma cruzi, the causative agent of Chagas disease.

    Science.gov (United States)

    Pan, Peiwen; Vermelho, Alane Beatriz; Capaci Rodrigues, Giseli; Scozzafava, Andrea; Tolvanen, Martti E E; Parkkila, Seppo; Capasso, Clemente; Supuran, Claudiu T

    2013-02-28

    An α-carbonic anhydrase (CA, EC 4.2.1.1) has been identified, cloned, and characterized from the unicellular protozoan Trypanosoma cruzi, the causative agent of Chagas disease. The enzyme (TcCA) has a very high catalytic activity for the CO2 hydration reaction, being similar kinetically to the human (h) isoform hCA II, although it is devoid of the His64 proton shuttle. A large number of aromatic/heterocyclic sulfonamides and some 5-mercapto-1,3,4-thiadiazoles were investigated as TcCA inhibitors. The aromatic sulfonamides were weak inhibitors (K(I) values of 192 nM to 84 μM), whereas some heterocyclic compounds inhibited the enzyme with K(I) values in the range 61.6-93.6 nM. The thiols were the most potent in vitro inhibitors (K(I) values of 21.1-79.0 nM), and some of them also inhibited the epimastigotes growth of two T. cruzi strains in vivo.

  8. 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.

  9. Carbonic anhydrase inhibitors. Inhibition of human cytosolic isoforms I and II with (reduced) Schiff's bases incorporating sulfonamide, carboxylate and carboxymethyl moieties.

    Science.gov (United States)

    Nasr, Gihane; Cristian, Alina; Barboiu, Mihail; Vullo, Daniella; Winum, Jean-Yves; Supuran, Claudiu T

    2014-05-15

    A library of Schiff bases was synthesized by condensation of aromatic amines incorporating sulfonamide, carboxylic acid or carboxymethyl functionalities as Zn(2+)-binding groups, with aromatic aldehydes incorporating tert-butyl, hydroxy and/or methoxy groups. The corresponding amines were thereafter obtained by reduction of the imines. These compounds were assayed for the inhibition of two cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes, hCA I and II. The Ki values of the Schiff bases were in the range of 7.0-21,400nM against hCA II and of 52-8600nM against hCA I, respectively. The corresponding amines showed Ki values in the range of 8.6nM-5.3μM against hCA II, and of 18.7-251nM against hCA I, respectively. Unlike the imines, the reduced Schiff bases are stable to hydrolysis and several low-nanomolar inhibitors were detected, most of them incorporating sulfonamide groups. Some carboxylates also showed interesting CA inhibitory properties. Such hydrosoluble derivatives may show pharmacologic applications.

  10. Synthesis of 5-amino-1,3,4-thiadiazole-2-sulphonamide derivatives and their inhibition effects on human carbonic anhydrase isozymes.

    Science.gov (United States)

    Kasımoğulları, Rahmi; Bülbül, Metin; Mert, Samet; Güleryüz, Hülya

    2011-04-01

    In this study, some novel inhibitors were synthesised from the further stage reactions of 4-benzoyl-1-(4-nitrophenyl)-5-phenyl-1H-pyrazole-3-carbonyl chloride with 5-amino-1,3,4-thiadiazole-2-sulphonamide 1 (inhibitor 1). They were characterised by elemental and spectral (¹H NMR, ¹³C NMR, IR) analyses. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of inhibitor 1, acetazolamide (2) and the 11 newly synthesised amides (8-18) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (K(i)) were determined. The K(i) values for the new compounds (8-18) were observed to be well below that of the parent compound inhibitor 1 and were also compared to 2 under the same experimental conditions. The comparison of the newly synthesised amides to inhibitor 1 and to 2 indicated that the new derivatives preferentially inhibited hCA-II and were more potent inhibitors of hCA-II than the parent inhibitor 1 and 2.

  11. 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.

  12. 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.

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

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    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.

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

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    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.

  15. 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.

  16. 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.

  17. 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.

  18. 水化层影响酸酐酶内CO2扩散行为的分子动力学模拟%Molecular dynamics simulation for hydration effect on CO2 diffusion in carbonic anhydrase

    Institute of Scientific and Technical Information of China (English)

    陈功; 卢滇楠; 吴建中; 刘铮

    2015-01-01

    The hydration layer of the enzyme in the bulk gas phase has great effects on its catalytic performance. Molecular dynamics (MD) simulations at all-atom level was applied to investigate the effects of the hydration layer thickness on the diffusion of carbon dioxide molecules into the active site of a carbonic anhydrase enzyme from a bulk gas phase. Based on the distribution of water molecules surrounding the carbonic anhydrase enzyme, the effects of the hydration layer thickness on the protein structure and CO2 transport from the bulk gas phase to the protein active site was studied. The simulation results suggested an optimal hydration layer thickness of 0.7 nm for CO2 diffusion. The CO2 adsorption sites were identified, which compose of the diffusion channel inside the carbonic anhydrase. The MD simulation revealed the open states of these adsorption sites, which may be useful to identify the bottleneck position of the diffusion channel. The molecular insight is helpful for design and optimization of carbonic anhydrase, enabling more efficient CO2 adsorption and conversion.%气相中酶分子表面的水化层对其催化行为具有显著的影响。本文采用全原子分子动力学模拟方法考察了气相体系碳酸酐酶表面的水化层对酶结构以及CO2在酶分子中扩散行为的影响。首先展现了水分子在酶分子及其活性中心周围的分布,研究了水化层厚度对于酶结构以及CO2扩散速率的影响;发现最有利于CO2扩散进入酶分子的水化层厚度为0.7 nm。确认了碳酸酐酶内CO2的吸附位点,通过对其开合状态统计,显示出碳酸酐酶中CO2扩散通道中的瓶颈位置。上述结果对设计和优化碳酸酐酶催化气相体系中CO2的吸附和转化提供了依据和启示。

  19. Specificity of mutations induced by carbon ions in budding yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matuo, Youichirou [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Nishijima, Shigehiro [Graduate School of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871 (Japan); Hase, Yoshihiro [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Sakamoto, Ayako [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Tanaka, Atsushi [Radiation-Applied Biology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Watanuki-machi 1233, Takasaki, Gunma 370-1292 (Japan); Shimizu, Kikuo [Radioisotope Research Center, Osaka University, Yamada-oka 2-4, Suita, Osaka 565-0871 (Japan)]. E-mail: shimizu@rirc.osaka-u.ac.jp

    2006-12-01

    To investigate the nature of mutations induced by accelerated ions in eukaryotic cells, the effects of carbon-ion irradiation were compared with those of {gamma}-ray irradiation in the budding yeast Saccharomyces cerevisiae. The mutational effect and specificity of carbon-ion beams were studied in the URA3 gene of the yeast. Our experiments showed that the carbon ions generated more than 10 times the number of mutations induced by {gamma}-rays, and that the types of base changes induced by carbon ions include transversions (68.7%), transitions (13.7%) and deletions/insertions (17.6%). The transversions were mainly G:C {sup {yields}} T:A, and all the transitions were G:C {sup {yields}} A:T. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5'-AC(A/T)-3' sequence were found to be easily changed. Large deletions and duplications were not observed, whereas ion-induced mutations in Arabidopsis thaliana were mainly short deletions and rearrangements. The remarkable feature of yeast mutations induced by carbon ions was that the mutation sites were localized near the linker regions of nucleosomes, whereas mutations induced by {gamma}-ray irradiation were located uniformly throughout the gene.

  20. 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

  1. 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.

  2. Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes.

    Science.gov (United States)

    Musa-Aziz, Raif; Occhipinti, Rossana; Boron, Walter F

    2014-11-01

    Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3 (-) hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3 (-)/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pHi changes (τpHi ) and pHS relaxations (τpHS ). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3 (-) buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τpHS , indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane.

  3. Mitochondrial carbonic anhydrase CA VB: differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles.

    Science.gov (United States)

    Shah, G N; Hewett-Emmett, D; Grubb, J H; Migas, M C; Fleming, R E; Waheed, A; Sly, W S

    2000-02-15

    A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

  4. V-H+ -ATPase translocation during blood alkalosis in dogfish gills: interaction with carbonic anhydrase and involvement in the postfeeding alkaline tide.

    Science.gov (United States)

    Tresguerres, Martin; Parks, Scott K; Wood, Chris M; Goss, Greg G

    2007-05-01

    We investigated the involvement of carbonic anhydrase (CA) in mediating V-H(+)-ATPase translocation into the basolateral membrane in gills of alkalotic Squalus acanthias. Immunolabeling revealed that CA is localized in the same cells as V-H(+)-ATPase. Blood plasma from dogfish injected with acetazolamide [30 mg/kg at time (t) = 0 and 6 h] and infused with NaHCO(3) for 12 h (1,000 microeq.kg(-1).h(-1)) had significantly higher plasma HCO(3)(-) concentration than fish that were infused with NaHCO(3) alone (28.72 +/- 0.41 vs. 6.57 +/- 2.47 mmol/l, n = 3), whereas blood pH was similar in both treatments (8.03 +/- 0.11 vs. 8.04 +/- 0.11 pH units at t = 12 h). CA inhibition impaired V-H(+)-ATPase translocation into the basolateral membrane, as estimated from immunolabeled gill sections and Western blotting on gill cell membranes (0.24 +/- 0.08 vs. 1.00 +/- 0.28 arbitrary units, n = 3; P < 0.05). We investigated V-H(+)-ATPase translocation during a postfeeding alkalosis ("alkaline tide"). Gill samples were taken 24-26 h after dogfish were fed to satiety in a natural-like feeding regime. Immunolabeled gill sections revealed that V-H(+)-ATPase translocated to the basolateral membrane in the postfed fish. Confirming this result, V-H(+)-ATPase abundance was twofold higher in gill cell membranes of the postfed fish than in fasted fish (n = 4-5; P < 0.05). These results indicate that 1) intracellular H(+) or HCO(3)(-) produced by CA (and not blood pH or HCO(3)(-)) is likely the stimulus that triggers the V-H(+)-ATPase translocation into the basolateral membrane in alkalotic fish and 2) V-H(+)-ATPase translocation is important for enhanced HCO(3)(-) secretion during a naturally occurring postfeeding alkalosis.

  5. 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.

  6. Effect of egg turning and incubation time on carbonic anhydrase gene expression in the blastoderm of the Japanese quail (Coturnix c. japonica).

    Science.gov (United States)

    De Winter, P; Sugden, D; Baggott, G K

    2008-09-01

    1. The gene expression of carbonic anhydrase, a key enzyme for the production of sub-embryonic fluid (SEF), was assessed in turned and unturned eggs of the Japanese quail. The plasma membrane-associated isoforms CA IV, CA IX, CA XII, CA XIV, and the cytoplasmic isoform CA II, were investigated in the extra-embryonic tissue of the blastoderm and in embryonic blood. 2. Eggs were incubated at 37.6 degrees C, c.60% RH, and turned hourly (90 degrees ) or left unturned. From 48 to 96 h of incubation mRNA was extracted from blastoderm tissue, reverse-transcribed to cDNA and quantified by real-time qPCR using gene-specific primers. Blood collected at 96 h was processed identically. 3. Blastoderm CA IV gene expression increased with the period of incubation only in turned eggs, with maxima at 84 and 96 h of incubation. Only very low levels were found in blood. 4. Blastoderm CA II gene expression was greatest at 48 and 54 h of incubation, subsequently declining to much lower levels and unaffected by turning. Blood CA II gene expression was about 25-fold greater than in the blastoderm. 5. The expression of CA IX in the blastoderm was the highest of all isoforms, yet unaffected by turning. CA XII did not amplify and CA XIV was present at unquantifiable low levels. 6. It is concluded that only gene expression for CA IV is sensitive to egg turning, and that increased CA IV gene expression could account for the additional SEF mass found at 84 to 96 h of incubation in embryos of turned eggs.

  7. Identification of putative unfolding intermediates of the mutant His-107-tyr of human carbonic anhydrase II in a multidimensional property space.

    Science.gov (United States)

    Halder, Puspita; Taraphder, Srabani

    2016-06-01

    In this article, we develop an extensive search procedure of the multi-dimensional folding energy landscape of a protein. Our aim is to identify different classes of structures that have different aggregation propensities and catalytic activity. Following earlier studies by Daggett et al. [Jong, D. D.; Riley, R.: Alonso, D.O.: Dagett, V. J. Mol. Biol. 2002, 319, 229], a series of high temperature all-atom classical molecular simulation studies has been carried out to derive a multi-dimensional property space. Dynamical changes in these properties are then monitored by projecting them along a one-dimensional reaction coordinate, dmean . We have focused on the application of this method to partition a wide array of conformations of wild type human carbonic anhydrase II (HCA II) and its unstable mutant His-107-Tyr along dmean by sampling a 35-dimensional property space. The resultant partitioning not only reveals the distribution of conformations corresponding to stable structures of HCA II and its mutant, but also allows the monitoring of several partially unfolded and less stable conformations of the mutant. We have investigated the population of these conformations at different stages of unfolding and collected separate sets of structures that are widely separated in the property space. The dynamical diversity of these sets are examined in terms of the loading of their respective first principal component. The partially unfolded structures thus collected are qualitatively mapped on to the experimentally postulated light molten globule (MGL) and molten globule (MG) intermediates with distinct aggregation propensities and catalytic activities. Proteins 2016; 84:726-743. © 2016 Wiley Periodicals, Inc.

  8. 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.

  9. 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

  10. 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.

  11. Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism

    Science.gov (United States)

    Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

    2017-01-01

    Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis. PMID:28241048

  12. 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.

  13. Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

    Science.gov (United States)

    Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

    2008-03-01

    Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

  14. Effect of carbon source perturbations on transcriptional regulation of metabolic fluxes in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ülgen Kutlu Ö

    2007-03-01

    Full Text Available Abstract Background Control effective flux (CEF of a reaction is the weighted sum of all fluxes through that reaction, derived from elementary flux modes (EFM of a metabolic network. Change in CEFs under different environmental conditions has earlier been proven to be correlated with the corresponding changes in the transcriptome. Here we use this to investigate the degree of transcriptional regulation of fluxes in the metabolism of Saccharomyces cerevisiae. We do this by quantifying correlations between changes in CEFs and changes in transcript levels for shifts in carbon source, i.e. between the fermentative carbon source glucose and nonfermentative carbon sources like ethanol, acetate, and lactate. The CEF analysis is based on a simple stoichiometric model that includes reactions of the central carbon metabolism and the amino acid metabolism. Results The effect of the carbon shift on the metabolic fluxes was investigated for both batch and chemostat cultures. For growth on glucose in batch (respiro-fermentative cultures, EFMs with no by-product formation were removed from the analysis of the CEFs, whereas those including any by-products (ethanol, glycerol, acetate, succinate were omitted in the analysis of growth on glucose in chemostat (respiratory cultures. This resulted in improved correlations between CEF changes and transcript levels. A regression correlation coefficient of 0.60 was obtained between CEF changes and gene expression changes in the central carbon metabolism for the analysis of 5 different perturbations. Out of 45 data points there were no more than 6 data points deviating from the correlation. Additionally, up- or down-regulation of at least 75% of the genes were in qualitative agreement with the CEF changes for all perturbations studied. Conclusion The analysis indicates that changes in carbon source are associated with a high degree of hierarchical regulation of metabolic fluxes in the central carbon metabolism as the

  15. Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

    OpenAIRE

    Coschigano, P W; Miller, S. M; Magasanik, B

    1991-01-01

    We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This U...

  16. The L-arginine/NO pathway, homoarginine, and nitrite-dependent renal carbonic anhydrase activity in young people with type 1 diabetes mellitus.

    Science.gov (United States)

    Carmann, Christina; Lilienthal, Eggert; Weigt-Usinger, Katharina; Schmidt-Choudhury, Anjona; Hörster, Irina; Kayacelebi, Arslan Arinc; Beckmann, Bibiana; Chobanyan-Jürgens, Kristine; Tsikas, Dimitrios; Lücke, Thomas

    2015-09-01

    High circulating levels of asymmetric dimethylarginine (ADMA) and low circulating levels of homoarginine (hArg) are known cardiovascular risk factors in adults. While in adults with type 1 diabetes mellitus (T1DM) circulating ADMA is significantly elevated, in children and adolescents the reported ADMA data are contradictory. In 102 children with T1DM and 95 healthy controls (HC) serving as controls, we investigated the L-arginine (Arg)/nitric oxide (NO) pathway. Children with T1DM were divided into two groups, i.e., in children with newly diagnosed diabetes mellitus [T1DM-ND; n = 10; age, 8.8 (4.4-11.2) years; HbA1c, 13 (8.9-13.9) %] and in those with long-term treatment [T1DM-T; n = 92; age, 12.5 (10.5-15.4) years; HbA1c, 8.0 (7.2-8.6) %]. The age of the HC was 11.3 (8-13.3) years. Amino acids and NO metabolites of the Arg/NO pathway, creatinine and the oxidative stress biomarker malondialdehyde (MDA) were measured by GC-MS or GC-MS/MS. Plasma hArg, ADMA and the hArg/ADMA molar ratio did not differ between the T1DM and HC groups. There was a significant difference between T1DM-T and HC with regard to plasma nitrite [0.53 (0.48-0.61) vs 2.05 (0.86-2.36) µM, P 86-2.36) µM, P < 0.0001]. Plasma MDA did not differ between the groups. The urinary nitrate-to-nitrite molar ratio (UNOXR), a measure of nitrite-dependent renal carbonic anhydrase (CA) activity, was higher in T1DM-T [1173 (738-1481), P < 0.0001] and T1DM-ND [1341 (1117-1615), P = 0.0007] compared to HC [540 (324-962)], but did not differ between T1DM-T and T1DM-ND (P = 0.272). The lower nitrite excretion in the children with T1DM may indicate enhanced renal CA-dependent nitrite reabsorption compared with healthy children. Yet, lower plasma nitrite concentration in the T1DM patients may have also contributed to the higher UNOXR. Patients' age correlated positively with plasma hArg and hArg/ADMA and urinary DMA/ADMA. Plasma ADMA and urinary ADMA, DMA, nitrite and nitrate correlated negatively with age of the

  17. 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

  18. 3D QSAR studies, pharmacophore modeling, and virtual screening of diarylpyrazole-benzenesulfonamide derivatives as a template to obtain new inhibitors, using human carbonic anhydrase II as a model protein.

    Science.gov (United States)

    Entezari Heravi, Yeganeh; Sereshti, Hassan; Saboury, Ali Akbar; Ghasemi, Jahan; Amirmostofian, Marzieh; Supuran, Claudiu T

    2017-12-01

    A 3D-QSAR modeling was performed on a series of diarylpyrazole-benzenesulfonamide derivatives acting as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The compounds were collected from two datasets with the same scaffold, and utilized as a template for a new pharmacophore model to screen the ZINC database of commercially available derivatives. The datasets were divided into training, test, and validation sets. As the first step, comparative molecular field analysis (CoMFA), CoMFA region focusing and comparative molecular similarity indices analysis (CoMSIA) in parallel with docking studies were applied to a set of 41 human (h) CA II inhibitors. The validity and the prediction capacity of the resulting models were evaluated by leave-one-out (LOO) cross-validation approach. The reliability of the model for the prediction of possibly new CA inhibitors was also tested.

  19. Comparison of QSAR models based on combinations of genetic algorithm, stepwise multiple linear regression, and artificial neural network methods to predict Kd of some derivatives of aromatic sulfonamides as carbonic anhydrase II inhibitors.

    Science.gov (United States)

    Maleki, Afshin; Daraei, Hiua; Alaei, Loghman; Faraji, Aram

    2014-01-01

    Four stepwise multiple linear regressions (SMLR) and a genetic algorithm (GA) based multiple linear regressions (MLR), together with artificial neural network (ANN) models, were applied for quantitative structure-activity relationship (QSAR) modeling of dissociation constants (Kd) of 62 arylsulfonamide (ArSA) derivatives as human carbonic anhydrase II (HCA II) inhibitors. The best subsets of molecular descriptors were selected by SMLR and GA-MLR methods. These selected variables were used to generate MLR and ANN models. The predictability power of models was examined by an external test set and cross validation. In addition, some tests were done to examine other aspects of the models. The results show that for certain purposes GA-MLR is better than SMLR and for others, ANN overcomes MLR models.

  20. 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.

  1. Characterization of one Novel Flavone and four New Source Compounds from the Bark of Millettia ovalifolia and In-Vitro Inhibition of Carbonic Anhydrase-II by the Novel Flavonoid

    Directory of Open Access Journals (Sweden)

    Taj Ur Rahman

    2015-06-01

    Full Text Available The phytochemical examination of the extract of bark of Millettia ovalifolia yielded chemical constituents, which included one novel flavonoid 7-(4-methoxyphenyl-9H-furo [2, 3-f] chromen-9-one and four new source compounds characterized as 3,7-Dihydroxy-2-phenyl-4H-chromen-4-one, (E-Ethyl-13-(3,4-dimethoxyphenylacrylate, (E-Methyl-3-(3,4-dimethoxyphenylacrylate and N-Ethylacetamide. These compounds were characterized by using advance modern spectroscopic analytical techniques such as UV, IR, 1D, 2D NMR and mass spectrometry. The novel flavonoid (1 displayed significant inhibition of cytosolic form of bovine carbonic anhydrase-II with IC 50 value of 17.86 ± 0.07 µM. This flavonoid may be used as a new pharmacophore to treat cystic fibrosis, glaucoma, epilepsy, leaukomia and other disorders such as neurology etc.

  2. Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose

    Directory of Open Access Journals (Sweden)

    Bergdahl Basti

    2012-05-01

    Full Text Available Abstract Background The concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR and xylitol dehydrogenase (XDH or the isomerization pathway with xylose isomerase (XI. The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production. Results Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool. Conclusions The low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH

  3. 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

  4. 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.

  5. 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

  6. Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

    Science.gov (United States)

    Coschigano, P W; Miller, S M; Magasanik, B

    1991-09-01

    We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

  7. Synthesis, characterization, computational studies, antimicrobial activities and carbonic anhydrase inhibitor effects of 2-hydroxy acetophenone-N-methyl p-toluenesulfonylhydrazone and its Co(II), Pd(II), Pt(II) complexes

    Science.gov (United States)

    Özbek, Neslihan; Alyar, Saliha; Memmi, Burcu Koçak; Gündüzalp, Ayla Balaban; Bahçeci, Zafer; Alyar, Hamit

    2017-01-01

    2-Hydroxyacetophenone-N-methyl p-toluenesulfonylhydrazone (afptsmh) derived from p-toluenesulfonicacid-1-methylhydrazide (ptsmh) and its Co(II), Pd(II), Pt(II) complexes were synthesized for the first time. Synthesized compounds were characterized by spectroscopic methods (FT-IR, 1Hsbnd 13C NMR, LC-MS, UV-vis), magnetic susceptibility and conductivity measurements. 1H and 13C shielding tensors for crystal structure of ligand were calculated with GIAO/DFT/B3LYP/6-311++G(d,p) methods in CDCl3. The vibrational band assignments were performed at B3LYP/6-311++G(d,p) theory level combined with scaled quantum mechanics force field (SQMFF) methodology. The antibacterial activities of synthesized compounds were studied against some Gram positive and Gram negative bacteria by using microdilution and disc diffusion methods. In vitro enzyme inhibitory effects of the compounds were measured by UV-vis spectrophotometer. The enzyme activities against human carbonic anhydrase II (hCA II) were evaluated as IC50 (the half maximal inhibitory concentration) values. It was found that afptsmh and its metal complexes have inhibitory effects on hCA II isoenzyme. General esterase activities were determined using alpha and beta naphtyl acetate substrates (α- and β-NAs) of Drosophila melanogaster (D. melanogaster). Activity results show that afptsmh does not strongly affect the bacteria strains and also shows poor inhibitory activity against hCAII isoenzyme whereas all complexes posses higher biological activities.

  8. The synthesis of novel pyrazole-3,4-dicarboxamides bearing 5-amino-1,3,4-thiadiazole-2-sulfonamide moiety with effective inhibitory activity against the isoforms of human cytosolic carbonic anhydrase I and II.

    Science.gov (United States)

    Mert, Samet; Alım, Zuhal; İşgör, Mehmet Mustafa; Beydemir, Şükrü; Kasımoğulları, Rahmi

    2016-10-01

    A series of 1-(3-substituted-phenyl)-5-phenyl-N(3),N(4)-bis(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-1H-pyrazole-3,4-dicarboxamides (4-15) were synthesized. The structures of these pyrazole-sulfonamides were confirmed by FT-IR, (1)H NMR, (13)C NMR and elemental analysis methods. Human cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozymes (hCA I and II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of newly synthesized derivatives (4-15) were investigated in vitro on esterase activities of these isozymes. The Ki values were determined as 0.119-3.999μM for hCA I and 0.084-0.878μM for hCA II. The results showed that the compound 6 for hCA I and the compound 11 for hCA II had the highest inhibitory effect. Beside that, the compound 8 had the lowest inhibition effect on both isozymes.

  9. Coordinated regulation of ammonium assimilation and carbon catabolism by glyoxylate in Saccharomyces cerevisiae.

    Science.gov (United States)

    González, A; Rodríguez, L; Folch, J; Soberón, M; Olivera, H

    1987-09-01

    The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.

  10. Changes of Carbonic Anhydrase Activities in Tomato Leaves under Drought Stress%干旱胁迫下番茄叶片碳酸酐酶活性的变化

    Institute of Scientific and Technical Information of China (English)

    孙卫红; 吴秋霞; 温新宇; 何华纲; 吴沿友; 丁佳俊; 封丽娜

    2015-01-01

    Carbonic anhydrase (CA) catalyzes the reversible hydration of CO2, and it is an important photosyn-thetic enzyme which take part in the CO2 conduction and get into the carboxylation site. The CA activity has a great effect on photosynthesis and water use efifciency. In order to study the effect of expression of CA gene on the plant photosynthesis and plant water potential in tomato (Lycopersicon esculentum), the expression of cytoso-lic CA gene in tomato leaves under drought stress were determined by Northern blotting. And the CA enzyme ac-tivity, photosynthetic rate and water potential in tomato leaves under drought stress were also determined. The re-sults showed that the expression of cytosolic CA gene in tomato leaves was induced by drought stress. With the time of drought stress, the CA activity increased and then fell, and the changes were basically similar to that of CA gene expression. Net photosynthetic rate and water potential of tomato leaves decreased with the degree of drought and the treatment time. However, the CA activity, net photosynthetic rate and water potential changed slightly with the soil relative water content of 75%. The results indicated that there was a certain corresponding relationship be-tween the changes of water potential and photosynthetic rate in tomato leaves and the change of CA activity.%碳酸酐酶(carbonic anhydrase, CA)催化CO2的可逆水合反应,是参与CO2传导而进入羧化位点的重要光合酶,其活性高低对植物水分利用及光合作用有较大影响。为了研究干旱胁迫下番茄CA表达活性变化对植株光合作用及水分利用的影响,本文利用Northern杂交检测番茄叶片的胞质CA基因在干旱胁迫下的表达;测定干旱胁迫下番茄叶片CA活性、光合速率和叶片水势变化。结果表明,番茄叶片胞质CA基因受干旱胁迫诱导;CA活性随着干旱胁迫时间延长而发生由低至高再回落的变化,且变化规律基本与CA基因相似。番

  11. 碳酸酐酶抑制剂乙酰唑胺对切口痛大鼠痛行为的影响%Effects of carbonic anhydrase inhibitor acetazolamide on pain behavior in a rat model of incision pain

    Institute of Scientific and Technical Information of China (English)

    韩潞潞; 赵华平; 薛庆生; 于布为

    2011-01-01

    Aim To observe the effect of carbonic anhydrase inhibitor acetazolamide ( ACT ) on the behavior of rats with incision pain. Methods All rats were intrathecally cathetered, six days later they were randomly divided into 5 groups with 16 rats in each group:sham, sham + ACT, incision pain, incision pain + low dose of ACT( 2.25 μg ), incision pain + high dose of ACT( 22.5 μg ). Rat' s incision pain model was established by using Brennan's method. ACT or vehicle was administered intrathecally 24 h after operation. Thermal withdrawal latency( TWL ) and mechanical withdrawal threshold( MWT ) were measured and compared 1 d before operation( baseline )and l d after operation( before drug injection and 30,75,120,165,240 min after drug injection ). Results The TWL and MWT were decreased significantly on 1 d after incision compared with baseline( P < 0.05 ). Intrathecal administration of highdose ACT increased the TWL at 30, 75 and 120 min after drug injection( P < 0.05 ). However, high dose ACT had no effect on the MWT. The TWL in incision + HACT group was significantly higher than that in incision group at 30, 75 and 120 min after drug injection ( P < 0.05 ). Conclusions ACT at large dose can partially alleviate incision-induced heat hyperalgesia but has no effect on the mechanical hyperalgesia. Carbonic anhydrase might be involved in the development of heat hyperalgesia induced by incision.%目的 观察鞘内注射碳酸酐酶抑制剂乙酰唑胺(ACT)对大鼠切口痛行为的影响.方法 所有大鼠术前6天鞘内置管,随机分为5组:假手术组、假手术+ACT组、切口痛组、切口痛+ACT低剂量(2.25 μg)组、切口痛+ACT高剂量(22.5 μg)组,每组16只.按照Brennan法建立切口痛模型.ACT和生理盐水均在术后d 1鞘内给予.分别于术前d 1(基础值)、术后d 1(给药前,给药后30、75、120、165、240 min)测定大鼠的热缩足潜伏期(TWL)和机械缩足反射阈值(MWT),并予比较.结果 切口痛术后d 1(给药

  12. Production of D-lactic acid in a continuous membrane integrated fermentation reactor by genetically modified Saccharomyces cerevisiae: enhancement in D-lactic acid carbon yield.

    Science.gov (United States)

    Mimitsuka, Takashi; Sawai, Kenji; Kobayashi, Koji; Tsukada, Takeshi; Takeuchi, Norihiro; Yamada, Katsushige; Ogino, Hiroyasu; Yonehara, Tetsu

    2015-01-01

    Poly d-lactic acid is an important polymer because it improves the thermostability of poly l-lactic acid by stereo complex formation. To demonstrate potency of continuous fermentation using a membrane-integrated fermentation reactor (MFR) system, continuous fermentation using genetically modified Saccharomyces cerevisiae which produces d-lactic acid was performed at the low pH and microaerobic conditions. d-Lactic acid continuous fermentation using the MFR system by genetically modified yeast increased production rate by 11-fold compared with batch fermentation. In addition, the carbon yield of d-lactic acid in continuous fermentation was improved to 74.6 ± 2.3% compared to 39.0 ± 1.7% with batch fermentation. This dramatic improvement in carbon yield could not be explained by a reduction in carbon consumption to form cells compared to batch fermentation. Further detailed analysis at batch fermentation revealed that the carbon yield increased to 76.8% at late stationary phase. S. cerevisiae, which exhibits the Crabtree-positive effect, demonstrated significant changes in metabolic activities at low sugar concentrations (Rossignol et al., Yeast, 20, 1369-1385, 2003). Moreover, lactate-producing S. cerevisiae requires ATP supplied not only from the glycolytic pathway but also from the TCA cycle (van Maris et al., Appl. Environ. Microbiol., 70, 2898-2905, 2004). Our finding was revealed that continuous fermentation, which can maintain the conditions of both a low sugar concentration and air supply, results in Crabtree-positive and lactate-producing S. cerevisiae for suitable conditions of d-lactic acid production with respect to redox balance and ATP generation because of releasing the yeast from the Crabtree effect.

  13. A Carbonic Anhydrase Serves as an Important Acid-Base Regulator in Pacific Oyster Crassostrea gigas Exposed to Elevated CO2: Implication for Physiological Responses of Mollusk to Ocean Acidification.

    Science.gov (United States)

    Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Qiu, Limei; Wang, Lingling; Zhang, Anguo; Song, Linsheng

    2017-02-01

    Carbonic anhydrases (CAs) have been demonstrated to play an important role in acid-base regulation in vertebrates. However, the classification and modulatory function of CAs in marine invertebrates, especially their responses to ocean acidification remain largely unknown. Here, a cytosolic α-CA (designated as CgCAII-1) was characterized from Pacific oyster Crassostrea gigas and its molecular activities against CO2 exposure were investigated. CgCAII-1 possessed a conserved CA catalytic domain, with high similarity to invertebrate cytoplasmic or mitochondrial α-CAs. Recombinant CgCAII-1 could convert CO2 to HCO3(-) with calculated activity as 0.54 × 10(3) U/mg, which could be inhibited by acetazolamide (AZ). The mRNA transcripts of CgCAII-1 in muscle, mantle, hepatopancreas, gill, and hemocytes increased significantly after exposure to elevated CO2. CgCAII-1 could interact with the hemocyte membrane proteins and the distribution of CgCAII-1 protein became more concentrated and dense in gill and mantle under CO2 exposure. The intracellular pH (pHi) of hemocytes under CO2 exposure increased significantly (p CO2 exposure. The impact of CO2 exposure on CA activity coupled with the mRNA expression level and protein translocation of CgCAII-1 provided evidences that CgCAII-1 could respond to ocean acidification and participate in acid-base regulation. Such cytoplasmic CA-based physiological regulation mechanism might explain other physiological responses of marine organisms to OA.

  14. 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.

  15. 意大利蜜蜂碳酸酐酶相关蛋白X的基因克隆及序列分析%Cloning and Sequence Analysis of Carbonic Anhydrase-Related Protein 10-like in Apis mellifera

    Institute of Scientific and Technical Information of China (English)

    李兆英

    2012-01-01

    [目的]对意大利蜜蜂(Apis mellifera)碳酸酐酶相关蛋白(CARPs)X的基因进行克隆,并对其序列进行分析.[方法]通过RTPCR扩增得到CARP X基因的cDNA序列,同时对序列进行生物信息学分析.[结果]意大利蜜蜂CARP X基因的cDNA全长972 bp,编码324个氨基酸残基,推导的氨基酸序列具有1个信号肽、2个跨膜区;预测其分子量和等电点分别为37.1 kD和7.458;该蛋白质与小蜜蜂(Apis florea)、熊蜂(Bombus impatiens)、Bombus terrestris、金小蜂(Nasonia vitripennis)、豌豆蚜虫(Acyrthosiphon pisum)的CARP X有很近的亲缘关系;聚类分析显示在昆虫体内可能具有2种不同类型的CARP X.[结论]该研究为CARPs蛋白家族的研究提供了可靠的理论基础.%[Objective] This study aimed to clone and analyze the gene sequence encoding carbonic anhydrase-related protein 10-like from Apis mellifera. [Method] The cDNA sequence of CARP X gene was cloned through RT-PCR method, and then analyzed with bioinformatical method. [Result] The full cDNA sequence of CARP X was 972 bp long, encoding 324 amino acid residues, including a signal peptide and two transmembrane domains. The predicted molecular mass was 37.1 kDa and the predicted isoelectric point was 7.458. The CARP X from A. mellifera showed the close relationship with proteins from Apis florae, Bombus impatiens, Bombus terrestris, Nasonia vitripennis and Acyrthosiphon pisum. The insect CARP X family may contain two suhfamilies. [Conclusion] The results provided the basis for studying CARPs family.

  16. 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

  17. The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

    OpenAIRE

    Dang, V D; Bohn, C.; Bolotin-Fukuhara, M; Daignan-Fornier, B.

    1996-01-01

    In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on a...

  18. Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bojsen, Rasmus K; Andersen, Kaj Scherz; Regenberg, Birgitte

    2012-01-01

    Microbial biofilms can be defined as multi-cellular aggregates adhering to a surface and embedded in an extracellular matrix (ECM). The nonpathogenic yeast, Saccharomyces cerevisiae, follows the common traits of microbial biofilms with cell-cell and cell-surface adhesion. S. cerevisiae is shown...... pathways including the protein kinase A and a mitogen-activated protein kinase pathway. Advanced genetic tools and resources have been developed for S. cerevisiae including a deletion mutant-strain collection in a biofilm-forming strain background and GFP-fusion protein collections. Furthermore, S....... cerevisiae biofilm is well applied for confocal laser scanning microscopy and fluorophore tagging of proteins, DNA and RNA. These techniques can be used to uncover the molecular mechanisms for biofilm development, drug resistance and for the study of molecular interactions, cell response to environmental...

  19. 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

  20. Effect of pH and light on population growth and activity of extracellular carbonic anhydrase in two species of dinoflagellates%pH及光照对两种赤潮甲藻种群生长和胞外碳酸酐酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    戴芳芳; 周成旭; 严小军

    2011-01-01

    研究了pH和光照强度对两种赤潮甲藻(Prorocentrum donghaiense和Karlodinium micrum)的种群生长及其胞外碳酸酐酶(CA)活性的影响.结果表明,pH7.5、光照强度30 μmol/(m2.s)是两种微藻的最适种群生长条件,在种群生长的最适pH和光照强度下,胞外CA活性最大.最适培养条件下,两种甲藻胞外碳酸酐酶活性随着种群生长周期而变化,指数生长期至平台期CA酶活增加,平台期至衰退期CA酶活降低,平台期胞外CA活性最大.%The effects of pH and light intensity on population growth and extracellular carbonic anhydrase ( CA) activity in two species of dinoflagellates (Prorocenlnim donghaieme and Karlodinium micrum) were studied. The results show that pH 7. 5 and 30μmol/( m2 · s) irradiance which triggered the optimum population growth, were the conditions that favored the highest activity of extracellular CA in the two dinoflagellates. Under the optimum cultivation conditions,the extracellular carbonic anhydrase activity in the two dinoflagellates varies with the population growth phases. CA activities were increased from exponential phase to the stationary phase and decreased form stationary phase to the decline phase. The highest extracellular CA activity occurred at the stationary phase. The significance of the population growth related CA activity in population dynamics of the two dinoflagellates was also discussed.

  1. Glucose repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kayikci, Omur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluc......Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration...... and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression...

  2. Studies on Saccharomyces cerevisiae under carbon-limiting growth transformed with plasmid pCYG4 that carries the gene for NADP-GDH.

    Science.gov (United States)

    Lima Filho, J L; Ledingham, W M

    1990-02-01

    The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).

  3. 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

  4. The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

    Science.gov (United States)

    Dang, V D; Bohn, C; Bolotin-Fukuhara, M; Daignan-Fornier, B

    1996-04-01

    In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.

  5. Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

    NARCIS (Netherlands)

    Verwaal, R.; Jiang, Y.; Wang, J.; Daran, J.M.; Sandmann, G.; Berg, van den J.A.; Ooyen, van A.J.J.

    2010-01-01

    To obtain insight into the genome-wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon-limited chemosta

  6. Effects of pentamidine isethionate on Saccharomyces cerevisiae.

    OpenAIRE

    Ludewig, G.; Williams, J M; Li, Y.; Staben, C

    1994-01-01

    We used Saccharomyces cerevisiae as a model system in which to examine the mechanism of action of the anti-Pneumocystis drug pentamidine. Pentamidine at low concentrations inhibited S. cerevisiae growth on nonfermentable carbon sources (50% inhibitory concentration [IC50] of 1.25 micrograms/ml in glycerol). Pentamidine inhibited growth on fermentable energy sources only at much higher concentrations (IC50 of 250 micrograms/ml in glucose). Inhibition at low pentamidine concentrations in glycer...

  7. One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, Vaibhavkumar N. [Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007 (India); Jha, Sanjay [Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, 395007 (India); Kailasa, Suresh Kumar, E-mail: sureshkumarchem@gmail.com [Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007 (India)

    2014-05-01

    We are reporting highly economical plant-based hydrothermal method for one-pot green synthesis of water-dispersible fluorescent carbon dots (CDs) by using Saccharum officinarum juice as precursor. The synthesized CDs were characterized by UV-visible, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high-resolution transmission electron microscopic (HR-TEM), and laser scanning confocal microscopic techniques. The CDs are well dispersed in water with an average size of ∼ 3 nm and showed bright blue fluorescence under UV-light (λ{sub ex} = 365 nm). These CDs acted as excellent fluorescent probes in cellular imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). - Highlights: • One-pot green synthesis was used for fluorescent CDs. • FT-IR, DLS, and TEM were used for the characterization of CDs. • The CDs are well dispersed in water with an average size of ∼ 3 nm. • The CDs acted as fluorescent probes for imaging of bacteria and yeast cells.

  8. 小新月菱形藻碳酸酐酶活性和光合作用对高盐度胁迫的响应%Response of carbonic anhydrase activity and photosynthesis to high salinity stress in Nitzschia closterium f.minutissima

    Institute of Scientific and Technical Information of China (English)

    余锦兰; 夏建荣; 邹永东

    2011-01-01

    Salinity is an important ecological factor in the algal growth.Water loss would lead to the increase of salinity in mass culture of fish food diatom, which could affect the algal photosynthesis and inorganic carbon utilization.In this paper, impacts of salinity on the growth, carbonic anhydrase activity, photosynthesis (P-I curve )and chlorophyll a fluorescence parameters in Nitzschia closterium f.minutissima were investigated to explore the photosynthetic mechanism of the diatom under the high salinity.The results showed that the specific growth rate when Nitzschia closterium f.minutissima was grown in high salinity (70)was decreased by 59.2% compared with the normal seawater, but it can maintain the growth to a certain degree in salinity up to 60 - 70, which suggested that Nitzschia closterium f.minutissima had a stronger tolerance to high salinity.The extracellular carbonic anhydrase activity which was measured by an electrometric method was reduced by 66.3%, and Chl.a and Chl.c contents which was determined spectrophotometrically were also decreased by 50.0% and 45.7% in high salinity (70).The extracellular CA activity was inhibited in high salinity environment which showed that capacity of HCO3- catalyzed by extracellular CA into CO2 declined, and high salinity stress can damage the Chl.a/b light harvesting complex (LHCII) and reaction-center complex and affect pigment synthesis.The chlorophyll a fluorescence parameters were measured by saturation pulse, and the maximal efficiency of PS Ⅱ photochemistry ( Fv/Fm ),actual photochemical efficiency of PS Ⅱ (Yield), photochemical quenching co-efficient (qP)decreased,whereas non-photochemical quenching co-efficient (qN)increased when the algae were grown in high salinity.These results showed that the high salinity stress would damage the PS Ⅱ reaction center, inhibit primary reaction of photosynthesis as well as the process of photosynthetic electron transport.The photosynthetic rate in the varied

  9. 碳酸酐酶Ⅸ(CA-Ⅸ)与HIF-1α在前列腺癌中的表达情况及相关性研究%The expression and correlation studies about Carbon anhydrase Ⅸ (CA-Ⅸ) and Hypoxia-inducible factor-1 alpha in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    黄海; 韩金利; 姚友生; 谢文练; 黄健; 卢振权; 杜涛; 林天歆; 许可慰; 董文; 毕良宽; 郭正辉; 江春

    2012-01-01

    目的 探讨碳酸酐酶Ⅸ(CA-Ⅸ)及缺氧诱导因子-1α (HIF-1α)在前列腺癌不同分期分级中的表达及其内在联系情况. 方法 采用免疫组织化学S-P法及Western-blot检测正常前列腺组织、前列腺癌组织以及前列腺癌细胞系PC-3、Lncap中CA-Ⅸ及HIF-1α的表达情况,结合临床资料进行统计分析,评价CA-Ⅸ及HIF-1α表达情况与前列腺组织癌变分化程度之间的关系,同时分析两者之间的相关性.结果 在正常前列腺组织中CA-Ⅸ及HIF-1α基本不表达,在前列腺癌组织石蜡切片中,HIF处于高表达,其表达情况与前列腺癌病理分级相关.低分化的前列腺癌组织中HIF-1α的表达量高于高分化的前列腺癌组织.CA-Ⅸ在前列腺癌组织中表达率为37.5%,高于正常组织,与肿瘤分化程度无关.CA-Ⅸ及HIF-1α在前列腺癌组织中的表达情况具有相关性.结论 CA-Ⅸ及HIF-1α与前列腺癌的发生成正相关,而且两者在前列腺癌组织中的表达具有相关性,同时提示了以缺氧诱导因子通路为基础的分子机制在前列腺癌的演进中起到一定的作用.%Objective To study the expression and correlation of Carbon anhydrase Ⅸ (CA-Ⅸ) and Hypoxia-inducible factor-1 alpha (HIF-1α) in prostate cancer.Methods The immunohistochemistry of S-P and western-blot were used to detect the expression of Carbon anhydrase Ⅸ (CA-Ⅸ) and Hypoxia-inducible factor-1 alpha (HIF-1α) in normal prostate tissue,prostate cancer tissue,and prostate cancer cell lines PC-3,Lncap.Combined with clinical data,the statistical analysis on the evaluation of CA-Ⅸ and HIF-1α expression and prostate tissue differentiation degree relationship was done and the correlation between the two factor was analysed.Results In normal prostate tissue,CA-Ⅸ and HIF-1α almost did not express,but in prostate cancer tissue paraffin section,HIF-1α was at a high expression, and its expression had relationship with pathological

  10. 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

  11. Relationship of carbonic anhydrase Ⅱgene polymorphism with primary open angle glaucoma genetic susceptibility%碳酸酐酶Ⅱ基因多态性与原发性开角型青光眼遗传易感性的关系

    Institute of Scientific and Technical Information of China (English)

    吴安黛; 叶锌铭; 高和香; 李俊; 赵晨

    2016-01-01

    Abstract•AIM:To analyze the relationship of carbonic anhydraseⅡ gene polymorphism with primary open angle glaucoma genetic susceptibility.•METHODS:From January 2012 to December 2014, 50 cases in our hospital for treatment of primary open angle glaucoma ( study group ) and 50 cases in outpatient department of our hospital for health examination ( control group ) were tested. The conventional elbow venous blood was taken.The use of polymerase chain reaction ( PCR ) and restriction fragment length polymorphism test were used to detect the characteristics of carbonic anhydraseⅡgene polymorphism.•RESULTS:The locus site of rs3758078 and rs10504813 of the two groups were in line with Hardy -Weinberg equilibrium law ( Hardy -Weinberg equilibrium ) .The results displayed in rs10504813 site, the difference of genotype frequencies between the two groups was not statistically significant ( P>0.05 ) .But the difference in allele frequencies between the two groups was statistical significance (P0.05 ) .After the carbonic anhydrase Ⅱ polymorphism haplotype analysis in the two groups, TAC ( Tracking area code of cell served by neighbor Enb) haplotype carriers appeared lawer risk of primary open-angle glaucoma.•CONCLUSION:There is certain relation between the polymorphism and the risk of carbonic anhydrase primary open -angle glaucoma, and rs3758078 locus balance may be the main reason for low risk; TAC haplotype carriers appear lower risk of primary open angle glaucoma.%目的:分析碳酸酐酶Ⅱ基因多态性与原发性开角型青光眼遗传易感性的关系。方法:选取2012-01/2014-12在丽水市人民医院进行诊治的原发性开角型青光眼患者(观察组)50例与在丽水市人民医院门诊部体检的健康人(对照组)50例进行试验观察,常规肘静脉取血,使用聚合酶链反应和限制性片段长度多态性技术测试碳酸酐酶Ⅱ基因多态性的特点。结果:两组患者在和rs10504813位

  12. 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。

  13. Pairwise comparison of {sup 89}Zr- and {sup 124}I-labeled cG250 based on positron emission tomography imaging and nonlinear immunokinetic modeling: in vivo carbonic anhydrase IX receptor binding and internalization in mouse xenografts of clear-cell renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Cheal, Sarah M.; Punzalan, Blesida; Doran, Michael G.; Osborne, Joseph R. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Evans, Michael J. [Memorial Sloan-Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, NY (United States); Lewis, Jason S. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Program in Molecular Pharmacology and Chemistry, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Radiochemistry and Imaging Sciences Service, New York, NY (United States); Zanzonico, Pat [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Therapy Service, New York, NY (United States); Memorial-Sloan Kettering Cancer Center, New York, NY (United States); Larson, Steven M. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Program in Molecular Pharmacology and Chemistry, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Therapy Service, New York, NY (United States)

    2014-05-15

    The PET tracer, {sup 124}I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for presurgical diagnosis of clear-cell renal cell carcinoma (ccRCC) (Divgi et al. in Lancet Oncol 8:304-310, 2007; Divgi et al. in J Clin Oncol 31:187-194, 2013). The radiometal {sup 89}Zr, however, may offer advantages as a surrogate PET nuclide over {sup 124}I in terms of greater tumor uptake and retention (Rice et al. in Semin Nucl Med 41:265-282, 2011). We have developed a nonlinear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between {sup 124}I-cG250 and {sup 89}Zr-cG250 using a human ccRCC xenograft tumor model in mice. We believe that this unique model better relates quantitative imaging data to the salient biological features of tumor antibody-antigen binding and turnover. We conducted experiments with {sup 89}Zr-cG250 and {sup 124}I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial PET imaging was performed in mice bearing subcutaneous ccRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumors were derived from the PET images using nonlinear compartmental modeling. The two tracers demonstrated virtually identical tumor cell binding and internalization but showed markedly different retentions in vitro. Superior PET images were obtained using {sup 89}Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous washout from normal tissues. Estimates of cG250/CAIX complex turnover were 1.35 - 5.51 x 10{sup 12} molecules per hour per gram of tumor (20 % of receptors internalized per hour), and the ratio of {sup 124}I/{sup 89}Zr atoms released per unit time by tumor was 17.5. Pairwise evaluation of {sup 89}Zr-cG250 and {sup

  14. 碳酸酐酶Ⅱ基因多态性与原发性开角型青光眼遗传易感性的关系%Relationship between polymorphisms of carbonic anhydrase Ⅱ gene and genetic predisposition of primary open angle glaucoma

    Institute of Scientific and Technical Information of China (English)

    王圆圆; 李国栋; 袁玲; 聂胜洁; 曾柏瑞

    2015-01-01

    目的 探讨碳酸酐酶Ⅱ(carbonic anhydrase,CAⅡ)基因多态性与原发性开角型青光眼(primary open angle glaucoma,POAG)遗传易感性的关系.方法 采集81例POAG患者(POAG组)和60位正常人群(对照组)静脉血,应用聚合酶链反应及限制性片段长度多态性技术检测受试对象CAⅡ基因多态性的分布情况.结果 rs10504813位点T等位基因频率分布两组间差异有统计学意义[x2=4.040、P=0.04,OR =0.613、95%CI=(0.380 ~0.989)];基因型频率分布在两组间差异无统计学意义(P=0.133).rs3758078位点及rs703位点基因型频率分布及等位基因频率分布在两组间差异均无统计学意义(均为P>0.05).构建8种单倍型,CAⅡ基因在两组间的单倍型分布差异具有统计学意义(P =0.042),TAC单倍型携带者患POAG风险显著降低[OR =0.145、95% CI=(0.021 ~0.980),P=0.022].结论 CAⅡ基因多态性与POAG发病风险有相关性,rs10504813位点的T等位基因可能是其保护性因素;TAC单倍型携带者患POAG风险显著降低.

  15. 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

  16. Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae.

    Science.gov (United States)

    Umemura, K; Atomi, H; Izuta, M; Kanai, T; Takeshita, S; Ueda, M; Tanaka, A

    1997-01-03

    We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was

  17. 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...

  18. 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

  19. Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

    DEFF Research Database (Denmark)

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R. H.;

    2012-01-01

    Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose...... sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon......-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling...

  20. Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

    Science.gov (United States)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

  1. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  2. Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bergman, Alexandra; Siewers, Verena; Nielsen, Jens;

    2016-01-01

    Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does...... C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some...... demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could...

  3. Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

    NARCIS (Netherlands)

    Boender, L.G.M.; Almering, M.J.H.; Dijk, M.; Van Maris, A.J.A.; De Winde, J.H.; Pronk, J.T.; Daran-Lapujade, P.

    2011-01-01

    Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuousl

  4. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation.

    Science.gov (United States)

    Domingues, Lucília; Guimarães, Pedro M R; Oliveira, Carla

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity.

  5. 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.

  6. Preparation of Fluorescent Carbon Dots and Its Cytotoxicity for Saccharomyce cerevisiae%荧光碳点的合成及对酿酒酵母的毒性研究

    Institute of Scientific and Technical Information of China (English)

    黄淮青; 曾萍; 韩宝福; 徐淑坤

    2012-01-01

    以葡萄糖为碳源采用溶剂热法合成了荧光碳点.紫外吸收光谱、荧光光谱以及透射电镜照片表明,所合成的荧光碳点发光性能优异,分散性好,且无团聚现象.荧光碳点原溶液出现浓度淬灭现象,稀释60倍情况下荧光最强.以酿酒酵母为模型生物,考察了不同生长时期(调整期、对数期早期、对数期中期)的酿酒酵母与不同浓度的荧光碳点共培养后的生长曲线.结果表明,即使荧光碳点浓度在27.75 mmol·L-1条件下也没有影响酵母菌的生长曲线,可认为基本没有细胞毒性.比较了相同荧光强度下的荧光碳点与CdTe量子点对酿酒酵母的细胞毒性,结果表明荧光碳点的毒性显著低于量子点的毒性.%Fluorescent carbon dots (CDs) were synthesized by a solvothermal method with glucose as the carbon source. UV absorption and fluorescence spectroscopy, and transmission electron microscopy (TEM) results showed that the synthesized CDs feature the excellent optical performance and high dispersion without agglomeration. The as-prepared CDs solution displayed a concentration quenching phenomenon and emitted the strongest fluorescence after being diluted 60 times. By using Saccharomyce cerevisiae yeast as a model organism, we investigated the dependence of yeast's growth on the concentration of fluorescent CDs at various growth periods (adjustment, initial and middle logarithmic phases). It is shown that there is no influence on yeast's growth curve even the concentration of fluorescent CDs as high as 27.75 mmol·L-1. The yeast cytotoxicity of fluorescent CDs and CdTe quantum dots were also compared under the same fluorescent intensity, indicating a much lower cytotoxicity fluorescent of CDs than that CdTe quantum dots.

  7. [Expression of inulinase genes in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus].

    Science.gov (United States)

    Sokolenko, G G; Karpechenko, N A

    2015-01-01

    Expression of the genes encoding the enzymes involved in inulin, sucrose, and glucose metabolism in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus was studied. The exon-intron structure of the relevant genes was identified and the primers for quantitative PCR were optimized. Expression of the genes was found to depend on the carbon source. Glucose was shown to exhibit a repressive effect on inulinase synthesis by K. marxianus, while in S. cerevisiae glucose and sucrose were inulinase inducer and repressor, respectively.

  8. Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory

    DEFF Research Database (Denmark)

    Otero, José Manuel; Cimini, Donatella; Patil, Kiran Raosaheb

    2013-01-01

    Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought......-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals....

  9. A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae

    Science.gov (United States)

    Economically viable production of lignocellulosic ethanol requires efficient conversion of feedstock sugars to ethanol. Saccharomyces cerevisiae cannot ferment xylose, the main five-carbon sugars in biomass, but can ferment xylulose, an enzymatically derived isomer. Xylulose fermentation is slow rel...

  10. Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation.

    Science.gov (United States)

    Dong, Shi-Jun; Lin, Xiang-Hua; Li, Hao

    2015-11-01

    During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.

  11. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    Energy Technology Data Exchange (ETDEWEB)

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. (Univ. di Milano (Italy))

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  12. levadura Saccharomyces Cerevisiae

    Directory of Open Access Journals (Sweden)

    B. Aguilar Uscanga

    2005-01-01

    Full Text Available La pared celular de levaduras representa entre 20 a 30 % de la célula en peso seco. Está compuesta de polisacáridos complejos de β-glucanos, manoproteínas y quitina. Se estudió la composición de los polisacáridos contenidos en la pared celular de la Saccharomyces cerevisiae CEN.PK 113 y se observó el efecto de la variación de la fuente carbono (glucosa, sacarosa, galactosa, maltosa, manosa, etanol y pH (3, 4, 5, 6 en un medio mineral “cell factory”. Las células fueron recolectadas en fase exponencial y se extrajo la pared celular. Los extractos de pared se hidrolizaron con H2SO4 al 72% y las muestras fueron analizadas por cromatografía HPLC. Se realizó una prueba de resistencia al rompimiento celular con una β(1,3-glucanasa, y las células cultivadas a diferentes fuentes carbono y pH. Los resultados del análisis por HPLC, mostraron que la composición de los polisacáridos en la pared celular, varía considerablemente con las modificaciones del medio de cultivo. Se observó que las levaduras cultivadas en sacarosa tienen mayor porcentaje de pared celular (25% y mayor cantidad de glucanos (115µg/mg peso seco y mananos (131µg/mg peso seco, que aquellas levaduras cultivadas en etanol (13% en peso seco. Las levaduras cultivadas a pH 5 presentaron 19% de pared celular en peso seco, mientras que a pH 6 el porcentaje fue menor (14%. El análisis de resistencia al rompimiento celular, mostró que las células cultivadas en etanol y galactosa fueron resistentes al rompimiento enzimático. Se comparó este resultado con el contenido de polisacáridos en la pared celular y concluimos que la resistencia de la célula al rompimiento, no está ligada con la cantidad de β-glucanos contenidos en la pared celular, sino que va a depender del número de enlaces β(1,3 y β(1,6-glucanos, los cuales juegan un rol importante durante el ensamblaje de la pared

  13. Proteomics of Saccharomyces cerevisiae Organelles

    NARCIS (Netherlands)

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them wi

  14. Fungal genomics beyond Saccharomyces cerevisiae?

    DEFF Research Database (Denmark)

    Hofmann, Gerald; Mcintyre, Mhairi; Nielsen, Jens

    2003-01-01

    Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious that the a......Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious...... that the application of the existing methods of genome, transcriptome, proteome and metabolome analysis to other fungi has enormous potential, especially for the production of food and food ingredients. The developments in the past year demonstrate that we have only just started to exploit this potential....

  15. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Silljé, H H; Paalman, J W; ter Schure, E G; Olsthoorn, S Q; Verkleij, A J; Boonstra, Johannes; Verrips, C T

    1999-01-01

    Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. B

  16. Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    de Kroon, A.I.P.M.

    2007-01-01

    Phosphatidylcholine (PC) is a very abundant membrane lipid in most eukaryotes including the model organism Saccharomyces cerevisiae. Consequently, the molecular species profile of PC, i.e. the ensemble of PC molecules with acyl chains differing in number of carbon atoms and double bonds, is importan

  17. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Miletti Luiz C

    2008-02-01

    Full Text Available Abstract Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by

  18. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    OpenAIRE

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic...

  19. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  20. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    Science.gov (United States)

    Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua

    2002-01-01

    Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

  1. Production of 1,2-propanediol from glycerol in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jung, Joon-Young; Yun, Hyun Shik; Lee, Jinwon; Oh, Min-Kyu

    2011-08-01

    Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.

  2. Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources Produção e caracterização da glucoamilase do fungo Aspergillus awamori expressa em levedura Saccharomyces cerevisiae usando diferentes fontes de carbono

    Directory of Open Access Journals (Sweden)

    Fabiana Carina Pavezzi

    2008-03-01

    Full Text Available Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55ºC, and, in the substratum absence, the thermostability was for 1h at 50ºC. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65ºC was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.A glucoamilase é amplamente utilizada na indústria de alimentos no processamento do amido para a produção de xarope com alto teor de glicose e também muito empregada nos processos de fermentação para produção de cerveja e etanol. Neste trabalho a glucoamilase de Aspergillus awamori expressa em Saccharomyces cerevisiae produzida sob fermentação líquida foi avaliada quanto à produtividade em diferentes amidos e caracterizada físico-quimicamente. A enzima apresentou alta atividade específica de 13,8 U/mg proteína e de 2,9 U/mg biomassa ao final de 48 h de fermentação em meio contendo amido solúvel. A glucoamilase apresentou temperatura ótima de atividade a 55ºC, e temperatura de desnaturação térmica na ausência de substrato por 1h a 50ºC. O pH ótimo de atividade foi na faixa de 3,5 - 4,0 e a estabilidade ao pH entre os valores 5,0 e 7,0. A meia vida a 65ºC foi 30,2 min., e a

  3. Influence of exopolymeric materials on bacterially induced mineralization of carbonates.

    Science.gov (United States)

    Bains, Amrita; Dhami, Navdeep Kaur; Mukherjee, Abhijit; Reddy, M Sudhakara

    2015-04-01

    Microbially induced calcium carbonate precipitation is an immensely growing technology for restoration and remediation of building materials. The investigation on role of exopolymeric substances and biofilms in microbially induced calcium carbonate precipitation suggested that these exopolymeric materials play major role in carbonate precipitation in Bacillus megaterium SS3 along with enzymes urease and carbonic anhydrase. The concentration of EPS directly affects the precipitation of carbonate precipitates which might be due to capturing of Ca(2+) ions by acting as nucleation site. Components of the media and presence of calcium also play crucial role in production of exopolymeric substances along with affecting the morphology of carbonate precipitates.

  4. Redox balancing in recombinant strains of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Anderlund, M.

    1998-09-01

    In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD{sup +} by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channeled metabolic transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was co expressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogenase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of

  5. Development Of An Efficient Glycerol Utilizing Saccharomyces Cerevisiae Strain Via Adaptive Laboratory Evolution

    DEFF Research Database (Denmark)

    Strucko, Tomas; Zirngibl, Katharina; Tharwat Tolba Mohamed, Elsayed

    2015-01-01

    With increasing interest in biosustainable technologies, the need for converting available non-saccharide carbon sources most efficiently is emerging. Highly abundant crude glycerol, a major waste residue in biodiesel production, has attracted attention as a cheap carbon source for microbial...... fermentation processes. The most commonly known microbial cell factory, the yeast Saccharomyces cerevisiae, has been extensively applied for the production of a wide range of scientifically and industrially relevant products using saccharides (mainly glucose) as carbon source. However, it was shown...

  6. Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2015-04-01

    Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway engineering and evolutionary engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast.

  7. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek;

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  8. 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.

  9. Prediction of activity of carbonic anhydrase inhibitor drugs based on QSAR studies

    Directory of Open Access Journals (Sweden)

    N. Darzi

    2015-06-01

    Full Text Available A quantitative structure-activity relationship (QSAR model, based on three quantum chemical descriptors obtained from the benzene sulphonamide derivatives using the density functional theory (DFT method. Then this developed model was used to predict the benzene sulphonamide binding constant. The QSAR model has correlation coefficient R of 0.901 and the standard error of 0.646. Also, the predictive power of this model was further examined by leave-7-out cross validation procedure which the obtained statistical parameters were: Q2= 0.991 and SPRESS= 0.4686 that giving a good enough predictive power. The selected descriptorsare: molecular weight (MW, absolute hardness (AH, HOMO energy (HOMO, respectively.

  10. 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.

  11. 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.

  12. Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration.

    NARCIS (Netherlands)

    Yang, Z.; Alvarez, B.V.; Chakarova, C.; Jiang, L.; Karan, G.; Frederick, J.M.; Zhao, Y.; Sauve, Y.; Li, X.; Zrenner, E.; Wissinger, B.; Hollander, A.I. den; Katz, B.; Baehr, W.; Cremers, F.P.M.; Casey, J.R.; Bhattacharya, S.S.; Zhang, K.

    2005-01-01

    Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown.

  13. Carbonic anhydrase-9 expression levels and prognosis in human breast cancer: association with treatment outcome.

    NARCIS (Netherlands)

    Span, P.N.; Bussink, J.; Manders, P.; Beex, L.V.A.M.; Sweep, C.G.J.

    2003-01-01

    Here, we set out to assess CA9 expression levels by real-time quantitative RT-PCR in breast cancer tissue samples obtained from 253 patients, and correlated those with relapse-free (RFS) survival. The median follow-up time was 75 months (range 2-168 months). CA9 expression was mainly found in high-g

  14. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

    Science.gov (United States)

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

  15. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.

    Science.gov (United States)

    Roca, Christophe; Nielsen, Jens; Olsson, Lisbeth

    2003-08-01

    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.

  16. Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

    DEFF Research Database (Denmark)

    Sonderegger, M.; Jeppsson, M.; Larsson, C.;

    2004-01-01

    Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrol......Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components...... in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation...

  17. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Penttilä Merja

    2008-06-01

    Full Text Available Abstract Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by

  18. Compositions and methods for modeling Saccharomyces cerevisiae metabolism

    DEFF Research Database (Denmark)

    2012-01-01

    The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S. cerevisiae reactants to a plurality of S. cerevisiae reactions, a constraint set for the plurality of S. cerevisiae reactions......, and commands for determining a distribution of flux through the reactions that is predictive of a S. cerevisiae physiological function. A model of the invention can further include a gene database containing information characterizing the associated gene or genes. The invention further provides methods...... for making an in silica S. cerevisiae model and methods for determining a S. cerevisiae physiological function using a model of the invention. The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S...

  19. EFFECT OF NITROGEN SOURCES ON THE PRODUCTION OF INVERTASE BY YEAST SACCHAROMYCES CEREVISIAE 3090

    OpenAIRE

    Suresh P. Kamble; Jyotsna C. Borate

    2012-01-01

    Invertase from Saccharomyces cerevisiae is high cost enzyme and primarily used in the confectionary industry. For large scale production of the enzyme, feasible synthetic medium with appropriate supplemented nutrients are required. The effect of carbon source on invertase production is well known, but little is known about the effect of different nitrogen source. The aim of the present study is to see the effect of different nitrogen sources on the production of invertase in submerged ferment...

  20. Multi-Capillary Column-Ion Mobility Spectrometry of Volatile Metabolites Emitted by Saccharomyces Cerevisiae

    OpenAIRE

    Christoph Halbfeld; Ebert, Birgitta E.; Blank, Lars M.

    2014-01-01

    Volatile organic compounds (VOCs) produced during microbial fermentations determine the flavor of fermented food and are of interest for the production of fragrances or food additives. However, the microbial synthesis of these compounds from simple carbon sources has not been well investigated so far. Here, we analyzed the headspace over glucose minimal salt medium cultures of Saccharomyces cerevisiae using multi-capillary column-ion mobility spectrometry (MCC-IMS). The high sensitivity and f...

  1. Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2016-03-01

    Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

  2. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

    2015-01-01

    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

  3. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  4. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  5. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Vázquez de Aldana Carlos R

    2008-10-01

    Full Text Available Abstract Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11 from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

  6. Characteristics of sterol uptake in Saccharomyces cerevisiae.

    OpenAIRE

    Lorenz, R T; Rodriguez, R J; Lewis, T A; Parks, L W

    1986-01-01

    A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were te...

  7. Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures.

    Science.gov (United States)

    Mendes, Filipa; Sieuwerts, Sander; de Hulster, Erik; Almering, Marinka J H; Luttik, Marijke A H; Pronk, Jack T; Smid, Eddy J; Bron, Peter A; Daran-Lapujade, Pascale

    2013-10-01

    Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.

  8. Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

    2014-10-01

    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.

  9. Flocculation of Saccharomyces cerevisiae tup1 mutants.

    OpenAIRE

    1984-01-01

    Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.

  10. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    Science.gov (United States)

    Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.

    2008-01-01

    Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…

  11. Nitrogen Catabolite Repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider

    1999-01-01

    In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Da180, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence S' GATAA 3'. Gln3...

  12. 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.

  13. New Perspectives on Acetate and One-Carbon Metabolism in the Methanoarchaea

    Energy Technology Data Exchange (ETDEWEB)

    Ferry, James [Pennsylvania State Univ., University Park, PA (United States)

    2017-03-20

    Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to bicarbonate. Although widespread in prokaryotes of the domains Bacteria and Archaea, few have been investigated and the physiological functions are largely unknown. Carbonic anhydrases are of biotechnological interest for carbon dioxide capture and sequestration at point sources. Prokaryotes encode three independently evolved classes. The alpha-class is restricted to a few pathogens and the other two are uniformly distributed in phylogenetically and physiologically diverse species. Although wide-spread in prokaryotes, only three gamma-class enzymes have been biochemically characterized and the physiological functions have not been investigated. The gamma-class is prominent in anaerobic acetate-utilizing methane-producing species of the genus Methanosarcina that encode three subclasses. Enzymes from two of the subclasses, Cam and CamH from Methanosarcina thermophila, have been characterized and found to utilize iron in the active site which is the first example of an iron-containing carbonic anhydrase. No representative of the third subclass has been isolated, although this subclass constitutes the great majority of the β-class. This grant application proposed to characterize gamma-class carbonic anhydrases from diverse anaerobic prokaryotes from the domains Bacteria and Archaea to broaden the understanding of this enzyme. In particular, the three subclasses present the genetically tractable acetate-utilizing methanogen Methanosarcina acetivorans will be investigated to extend studies of acetate and one-carbon metabolism in this species. A genetic approach will be taken to ascertain the physiological functions. It is also proposed to delve deeper into the mechanism of Cam from M. thermophila, the archetype of the gamma-class, via a high resolution neutron structure and kinetic analysis of site-specific amino acid replacement variants. In the course of the investigation, goals were added to

  14. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

    DEFF Research Database (Denmark)

    Qin, Jiufu; Zhou, Yongjin J.; Krivoruchko, Anastasia;

    2015-01-01

    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolite...... the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.......Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites...... of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway...

  15. 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.

  16. Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

    Science.gov (United States)

    Bharti, Randhir K; Srivastava, Shaili; Thakur, Indu Shekhar

    2014-01-01

    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.

  17. Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis.

    Science.gov (United States)

    Herwig, Christoph; Von Stockar, Urs

    2003-03-30

    A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess. However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response. The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S. kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model. It was found that, under transient growth conditions, S. kluyveri behaved like K. lactis, while classification using steady-state conditions would position S. kluyveri close to S. cerevisiae. For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S. cerevisiae, and carbon-driven, such as S. kluyveri and K. lactis, strains. Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents. The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively. The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose. The benefit from using small metabolic flux models is the richness of information and the

  18. Elucidating TOR Signaling and Rapamycin Action: Lessons from Saccharomyces cerevisiae

    OpenAIRE

    Crespo, José L; Hall, Michael N.

    2002-01-01

    TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding r...

  19. Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network

    DEFF Research Database (Denmark)

    Förster, Jochen; Famili, I.; Fu, P.;

    2003-01-01

    The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments...... containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to similar to16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared...

  20. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Elena Fossati

    Full Text Available Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S-reticuline starting from (R,S-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS, salutaridine reductase (PsSAR and salutaridinol acetyltransferase (PsSAT were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R-reticuline. Yeast cell feeding assays using (R-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.

  1. Saccharomyces cerevisiae metabolism in ecological context

    Science.gov (United States)

    Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R.

    2016-01-01

    The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype–phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype–environment–phenotype relationships. PMID:27634775

  2. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Vemuri, G. N.; Bao, X. M.;

    2009-01-01

    During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation....... However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect...... in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol...

  3. Terminal acidic shock inhibits sour beer bottle conditioning by Saccharomyces cerevisiae.

    Science.gov (United States)

    Rogers, Cody M; Veatch, Devon; Covey, Adam; Staton, Caleb; Bochman, Matthew L

    2016-08-01

    During beer fermentation, the brewer's yeast Saccharomyces cerevisiae experiences a variety of shifting growth conditions, culminating in a low-oxygen, low-nutrient, high-ethanol, acidic environment. In beers that are bottle conditioned (i.e., carbonated in the bottle by supplying yeast with a small amount of sugar to metabolize into CO2), the S. cerevisiae cells must overcome these stressors to perform the ultimate act in beer production. However, medium shock caused by any of these variables can slow, stall, or even kill the yeast, resulting in production delays and economic losses. Here, we describe a medium shock caused by high lactic acid levels in an American sour beer, which we refer to as "terminal acidic shock". Yeast exposed to this shock failed to bottle condition the beer, though they remained viable. The effects of low pH/high [lactic acid] conditions on the growth of six different brewing strains of S. cerevisiae were characterized, and we developed a method to adapt the yeast to growth in acidic beer, enabling proper bottle conditioning. Our findings will aid in the production of sour-style beers, a trending category in the American craft beer scene.

  4. Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges.

    Science.gov (United States)

    Abbott, Derek A; Zelle, Rintze M; Pronk, Jack T; van Maris, Antonius J A

    2009-12-01

    To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae, to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.

  5. Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

    Science.gov (United States)

    Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

    1998-01-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

  6. Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

    Science.gov (United States)

    Matsushika, Akinori; Hoshino, Tamotsu

    2015-12-01

    The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

  7. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    Science.gov (United States)

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  8. Social wasp intestines host the local phenotypic variability of Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Dapporto, Leonardo; Stefanini, Irene; Rivero, Damariz; Polsinelli, Mario; Capretti, Paolo; De Marchi, Paolo; Viola, Roberto; Turillazzi, Stefano; Cavalieri, Duccio

    2016-07-01

    Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess whether the strains found in wasp intestines are characterized by peculiar traits. We analysed strains isolated from the intestines of social wasps and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to representing a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains; their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved, to safeguard not only the natural variance of this microorganism but also the interests of wine-makers, who could take advantage from the exploitation of their phenotypic variability. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jared W Wenger

    2010-05-01

    Full Text Available Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

  10. Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

    2016-07-10

    In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without

  11. Investigation of autonomous cell cycle oscillation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hansen, Morten Skov

    2007-01-01

    Autonome Oscillationer i kontinuert kultivering af Saccharomyces cerevisiae Udgangspunktet for dette Ph.d. projekt var at søge at forstå, hvad der gør det muligt at opnå multiple statiske tilstande ved kontinuert kultivering af Saccharomyces cerevisiae med glukose som begrænsende substrat...

  12. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    Science.gov (United States)

    Raffar, Nur Izzati Abdul; Rahman, Nadhratul Nur Ain Abdul; Alrozi, Rasyidah; Senusi, Faraziehan; Chang, Siu Hua

    2015-05-01

    Biosorption of copper ion using treated and untreated immobilized Saccharomyces cerevisiae from aqueous solution was investigate in this study. S.cerevisiae has been choosing as biosorbent due to low cost, easy and continuously available from various industries. In this study, the ability of treated and untreated immobilized S.cerevisiae in removing copper ion influence by the effect of pH solution, and initial concentration of copper ion with contact time. Besides, adsorption isotherm and kinetic model also studied. The result indicated that the copper ion uptake on treated and untreated immobilized S.cerevisiae was increased with increasing of contact time and initial concentration of copper ion. The optimum pH for copper ion uptake on untreated and treated immobilized S.cerevisiae at 4 and 6. From the data obtained of copper ion uptake, the adsorption isotherm was fitted well by Freundlich model for treated immobilized S.cerevisiae and Langmuir model for untreated immobilized S.cerevisiae according to high correlation coefficient. Meanwhile, the pseudo second order was described as suitable model present according to high correlation coefficient. Since the application of biosorption process has been received more attention from numerous researchers as a potential process to be applied in the industry, future study will be conducted to investigate the potential of immobilized S.cerevisiae in continuous process.

  13. Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hansen, M.K.; Tams, J.W.; Fahrenkrug, Jan;

    1999-01-01

    G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide......G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide...

  14. Mechanisms of inorganic carbon acquisition in two estuarine Rhodophyceans: Bostrychia scorpioides (Hudson) ex Kützing Montagne and Catenella caespitosa (Withering) L. M. Irvine.

    Science.gov (United States)

    Ruiz-Nieto, Miriam; Fernández, José A; Niell, F Xavier; Carmona, Raquel

    2014-09-01

    Marine macroalgae possess a range of mechanisms to increase the availability of CO2 for fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase. Of these, possession of a periplasmic or external carbonic anhydrase and the ability to use bicarbonate ions is widely distributed. The mechanisms of carbon acquisition were studied in two estuarine red macroalgae Bostrychia scorpioides and Catenella caespitosa using a range of techniques. pH-drift and CO2-depletion experiments at constant pH suggested that CO2 is the main source of inorganic carbon in both species. Inhibitors indicated that internal and external carbonic anhydrase were present in both species. Inhibitors also suggested that uptake of bicarbonate is unlikely to be present (P < 0.05).

  15. In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains

    DEFF Research Database (Denmark)

    van der Aa Kuhle, Alis; Skovgaard, Kerstin; Jespersen, Lene

    2005-01-01

    strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar...

  16. Isocitrate lyase localisation in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Chaves, R S; Herrero, P; Ordiz, I; Angeles del Brio, M; Moreno, F

    1997-10-01

    The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.

  17. Systematic analysis of S. cerevisiae chromosome VIII genes.

    Science.gov (United States)

    Niedenthal, R; Riles, L; Güldener, U; Klein, S; Johnston, M; Hegemann, J H

    1999-12-01

    To begin genome-wide functional analysis, we analysed the consequences of deleting each of the 265 genes of chromosome VIII of Saccharomyces cerevisiae. For 33% of the deletion strains a growth phenotype could be detected: 18% of the genes are essential for growth on complete glucose medium, and 15% grow significantly more slowly than the wild-type strain or exhibit a conditional phenotype when incubated under one of 20 different growth conditions. Two-thirds of the mutants that exhibit conditional phenotypes are pleiotropic; about one-third of the mutants exhibit only one phenotype. We also measured the level of expression directed by the promoter of each gene. About half of the promoters direct detectable transcription in rich glucose medium, and most of these exhibited only low or medium activity. Only 1% of the genes are expressed at about the same level as ACT1. The number of active promoters increased to 76% upon growth on a non-fermentable carbon source, and to 93% in minimal glucose medium. The majority of promoters fluctuated in strength, depending on the medium.

  18. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions.

  19. Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing.

    Science.gov (United States)

    Fujihara, Hidehiko; Hino, Mika; Takashita, Hideharu; Kajiwara, Yasuhiro; Okamoto, Keiko; Furukawa, Kensuke

    2014-01-01

    We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.

  20. Response of Saccharomyces cerevisiae to cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Luciana Mara Costa; Ribeiro, Frederico Haddad; Neves, Maria Jose [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia], e-mail: luamatu@uol.com.br; Porto, Barbara Abranches Araujo; Amaral, Angela M.; Menezes, Maria Angela B.C. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Lab. de Ativacao Neutronica], e-mail: menezes@cdtn.br; Rosa, Carlos Augusto [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: carlrosa@icb.ufmg

    2009-07-01

    The intensification of industrial activity has been greatly contributing with the increase of heavy metals in the environment. Among these heavy metals, cadmium becomes a serious pervasive environmental pollutant. The cadmium is a heavy metal with no biological function, very toxic and carcinogenic at low concentrations. The toxicity of cadmium and several other metals can be mainly attributed to the multiplicity of coordination complexes and clusters that they can form. Some aspects of the cellular response to cadmium were extensively investigated in the yeast Saccharomyces cerevisiae. The primary site of interaction between many toxic metals and microbial cells is the plasma membrane. Plasma-membrane permeabilisation has been reported in a variety of microorganisms following cadmium exposure, and is considered one mechanism of cadmium toxicity in the yeast. In this work, using the yeast strain S. cerevisiae W303-WT, we have investigated the relationships between Cd uptake and release of cellular metal ions (K{sup +} and Na{sup +}) using neutron activation technique. The neutron activation was an easy, rapid and suitable technique for doing these metal determinations on yeast cells; was observed the change in morphology of the strains during the process of Cd accumulation, these alterations were observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) during incorporation of cadmium. (author)

  1. Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

    Directory of Open Access Journals (Sweden)

    Manuel Quirós

    Full Text Available As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.

  2. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Vemuri, Goutham; Eiteman, M.A; McEwen, J.E;

    2007-01-01

    Crabtree effect.’’ The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely...... effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation...... NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria....

  3. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    of translation of FLO11. In conclusion, I have conducted the first global study of the genetic program for yeast biofilm formation on polystyrene. This work provide several target genes as good basis for further research of biofilm, that I believe can contribute to fields such as cell biology, genetics, system......In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  4. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

  5. Viruses and prions of Saccharomyces cerevisiae.

    Science.gov (United States)

    Wickner, Reed B; Fujimura, Tsutomu; Esteban, Rosa

    2013-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for blocking the propagation of the viruses and prions, and proteins involved in the expression of viral components. Here, we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast.

  6. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars;

    2008-01-01

    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

  7. Glucose- and nitrogen sensing and regulatory mechanisms in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rødkaer, Steven V; Færgeman, Nils J.

    2014-01-01

    steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has...... been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae....

  8. Optimization of feeding strategy for the ergosterol production by yeasts Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mojmir Rychtera

    2010-08-01

    Full Text Available Objective of this study was to optimize ergosterol production by yeast strain Saccharomyces cerevisiae with the use of computer controlled feeding of cultivation medium. Baker´s yeasts strain of Saccharomyces cerevisiae originally modified and selected as mutant D7 was further applied in an industrial scale and also in this investigation. Composition of cultivation medium was optimized with the use of a modified Rosenbrock´s method with regard to following components: glucose, yeast extract, ammonium sulphate, potassium dihydrogen phosphate, magnesium sulphate and calcium chloride. Cultivation of yeast culture was performed in 7 L laboratory bioreactor with a working volume of 5 L equipped with a control unit and linked to a computer, with dissolved oxygen tension measurement, oxygen and carbon dioxide analyzers. BIOGENES prototype software was created from the commercial control system Genesis for Windows 3.0 (GFW, from Iconics and CLIPS 6.04 for the PC-Windows platform. From various factors affecting sterol biosynthesis a specific growth rate was chosen. Feed rate was controlled according to mathematical model. In this case it dealt with a design of optimal profile of specific growth rate with consequent calculation of carbon dioxide profile. Sterol concentration in the dry biomass increased from 1.0 % up to 3 %. Key words: Saccharomyces cerevisiae yeasts, ergosterol, fed-batch cultivation control, effect of the specific growth rate. Resumen: El objetivo de este estudio fue optimizar la producción de ergosterol por una cepa de levadura Saccharomyces cerevisiae, controlando la alimentación de medio de cultivo por computadora. La cepa de levadura panadera Saccharomyces cerevisiae originalmente modificada y seleccionada como mutante D7 fue posteriormente utilizada a escala industrial y también para esta investigación. La composición del medio de cultivo fue optimizada usando el método modificado de Rosenbrock respecto a los siguientes

  9. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  10. Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering

    Directory of Open Access Journals (Sweden)

    Ng Chiam Yu

    2012-05-01

    Full Text Available Abstract Background 2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production. Results We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1 and yield (0.113 g·g-1 were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition. Conclusions With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

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

    OpenAIRE

    2014-01-01

    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 product...

  12. Relationship among salivary carbonic anhydrase VI activity and flow rate, biofilm pH and caries in primary dentition

    OpenAIRE

    Fernanda Frasseto

    2011-01-01

    Resumo: Este estudo teve como objetivos determinar a atividade da anidrase carbônica VI (ACVI) na saliva de pré-escolares com cárie e investigar a relação entre a experiência cárie (dmfs) e a atividade da ACVI, o fluxo salivar e pH do biofilme antes e após o bochecho com sacarose a 20% em pré-escolares. Trinta pré-escolares com idade entre 45,3 e 80,3 meses foram divididos em 2 grupos: grupo livres de cárie (LC) e grupo com cárie (C). Exames clínicos foram realizados por um examinador de acor...

  13. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    Science.gov (United States)

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  14. PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Haiqing; Kim, Sooah; Sorek, Hagit; Lee, Youngsuk; Jeong, Deokyeol; Kim, Jungyeon; Oh, Eun Joong; Yun, Eun Ju; Wemmer, David E; Kim, Kyoung Heon; Kim, Soo Rin; Jin, Yong-Su

    2016-03-01

    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

  15. Metabolic flux analysis of Saccharomyces cerevisiae in a sealed winemaking fermentation system.

    Science.gov (United States)

    Li, Hua; Su, Jing; Ma, Wen; Guo, Anque; Shan, Zuhua; Wang, Hua

    2015-03-01

    A sealed fermentation (SF) system and an anaerobic fermentation (AF) system (under normal atmospheric pressure conditions) were employed to study the influence of endogenous carbon dioxide (CO2) on the metabolism of Saccharomyces cerevisiae. The results showed that the fermentation stopped when 82.0 g L(-1) glucose was consumed and the endogenously produced CO2: pressure reached to 14.3 MPa in SF system, while the sugar was used up during AF. The total yeast viable count in the end of AF was higher than that of SF. It was also observed that the ethanol yield in AF and SF was similar, the glycerol yield in AF was 1.26 times higher than that in SF, while the succinic acid and acetic acid yields in SF were 24.7 and 26 times higher than that in AF, respectively. Additionally, this work provides a stoichiometric model used for metabolic flux analysis of S. cerevisiae to compare the flux distribution in SF and AF. The results showed that CO2 had an important effect on the pathways of oxaloacetic acid formation from pyruvic acid and ribose-5-phosphate formation from glucose-6-phosphate. However, the pathway of ethanol formation from pyruvic acid (decarboxylation reaction), catalyzed by pyruvate decarboxylase, was insensitive to CO2.

  16. Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Kanchana R. Kildegaard

    2015-12-01

    Full Text Available Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP, a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L−1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol−1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose.

  17. Computational approaches for the genetic and phenotypic characterization of a Saccharomyces cerevisiae wine yeast collection.

    Science.gov (United States)

    Franco-Duarte, R; Umek, L; Zupan, B; Schuller, D

    2009-12-01

    Within this study, we have used a set of computational techniques to relate the genotypes and phenotypes of natural populations of Saccharomyces cerevisiae, using allelic information from 11 microsatellite loci and results from 24 phenotypic tests. A group of 103 strains was obtained from a larger S. cerevisiae winemaking strain collection by clustering with self-organizing maps. These strains were further characterized regarding their allelic combinations for 11 microsatellites and analysed in phenotypic screens that included taxonomic criteria (carbon and nitrogen assimilation tests, growth at different temperatures) and tests with biotechnological relevance (ethanol resistance, H(2)S or aromatic precursors formation). Phenotypic variability was rather high and each strain showed a unique phenotypic profile. The results, expressed as optical density (A(640)) after 22 h of growth, were in agreement with taxonomic data, although with some exceptions, since few strains were capable of consuming arabinose and ribose to a small extent. Based on microsatellite allelic information, naïve Bayesian classifier correctly assigned (AUC = 0.81, p 0.75). Subgroups were found for strains with low ethanol resistance, growth at 30 degrees C and growth in media containing galactose, raffinose or urea. The results demonstrate that computational approaches can be used to establish genotype-phenotype relations and to make predictions about a strain's biotechnological potential.

  18. Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae.

    Science.gov (United States)

    ter Schure, E G; Silljé, H H; Raeven, L J; Boonstra, J; Verkleij, A J; Verrips, C T

    1995-05-01

    Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.

  19. NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles.

    Science.gov (United States)

    DeLuna, A; Avendano, A; Riego, L; Gonzalez, A

    2001-11-23

    In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.

  20. Oxygen availability strongly affects chronological lifespan and thermotolerance in batch cultures of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Markus M.M. Bisschops

    2015-10-01

    Full Text Available Stationary-phase (SP batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability.

  1. A novel approach for the improvement of ethanol fermentation by Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Hou, L.; Cao, X.; Wang, C. [Tianjin Univ. of Science and Technology, Tianjin (China). Key Laboratory of Food Nutrition and Safety

    2010-06-15

    The partial substitution of fossil fuels with bioethanol has become an important strategy for the use of renewable energy. Ethanol production is generally achieved through fermentation of starch or sugar-based feedstock by Saccharomyces cerevisiae. In order to meet the growing demand for ethanol, there is a need for new yeast strains that can produce ethanol more efficiently and cost effectively. This paper presented a new genome engineering approach that was developed to improve ethanol production by S. cerevisiae. In this study, the aneuploid strain constructed on the base of tetraploid cells was shown to have favourable metabolic traits in very high gravity (VHG) fermentation with 300 g/L glucose as the carbon source. The tetraploid strain was constructed using the plasmid YCplac33-GHK, which comprised the HO gene encoding the site-specific HO endonucleases. The aneuploid strain, WT4-M, was chosen and screened once the tetraploid cells were treated with methyl benzimidazole-2-yl-carbamate to induce loss of mitotic chromosomes. The aneuploid strain WT4-M increased ethanol production as well as osmotic and thermal tolerance. The sugar to ethanol conversion rate also improved. It was concluded that this new approach is valuable for creating yeast strains with better fermentation characteristics. 25 refs., 3 figs.

  2. Fermentation of xylose to produce ethanol by recombinant Saccharomyces cerevisiae strain containing XYLA and XKS1

    Institute of Scientific and Technical Information of China (English)

    LIU Xiaolin; JIANG Ning; HE Peng; LU Dajun; SHEN An

    2005-01-01

    Fermentation of the pentose sugar xylose to produce ethanol using lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyce cerevisise, an efficient ethanol producer, cannot utilize xylose because it lacks the ability to convert xylose to its isomer xylulose. In this study, XYLA gene encoding xylose isomerase (XI) from Thermoanaerobacter tengcongensis MB4T and XKS1 gene encoding xylulokinase (XK) from Pichia stipitis were cloned and functionally coexpressed in Saccharomyces cerevisiae EF-326 to construct a recombinant xylose-utilizing strain. The resulting strain S. cerevisiae EF 1014 not only grew on xylose as sole carbon source, but also produced ethanol under anaerobic conditions. Fermentations performed with different xylose concentrations at different temperatures demonstrated that the highest ethanol productivity was 0.11 g/g xylose when xylose concentration was provided at 50 g/L. Under this condition, 28.4% of xylose was consumed and 1.54 g/L xylitol was formed. An increasing fermentation temperature from 30℃ to 37℃ did not improve ethanol yield.

  3. Multi-Capillary Column-Ion Mobility Spectrometry of Volatile Metabolites Emitted by Saccharomyces Cerevisiae

    Directory of Open Access Journals (Sweden)

    Christoph Halbfeld

    2014-09-01

    Full Text Available Volatile organic compounds (VOCs produced during microbial fermentations determine the flavor of fermented food and are of interest for the production of fragrances or food additives. However, the microbial synthesis of these compounds from simple carbon sources has not been well investigated so far. Here, we analyzed the headspace over glucose minimal salt medium cultures of Saccharomyces cerevisiae using multi-capillary column-ion mobility spectrometry (MCC-IMS. The high sensitivity and fast data acquisition of the MCC-IMS enabled online analysis of the fermentation off-gas and 19 specific signals were determined. To four of these volatile compounds, we could assign the metabolites ethanol, 2-pentanone, isobutyric acid, and 2,3-hexanedione by MCC-IMS measurements of pure standards and cross validation with thermal desorption–gas chromatography-mass spectrometry measurements. Despite the huge biochemical knowledge of the biochemistry of the model organism S. cerevisiae, only the biosynthetic pathways for ethanol and isobutyric acid are fully understood, demonstrating the considerable lack of research of volatile metabolites. As monitoring of VOCs produced during microbial fermentations can give valuable insight into the metabolic state of the organism, fast and non-invasive MCC-IMS analyses provide valuable data for process control.

  4. Changes of trehalose content and expression of relative genes during the bioethanol fermentation by Saccharomyces cerevisiae.

    Science.gov (United States)

    Yi, Chenfeng; Wang, Fenglian; Dong, Shijun; Li, Hao

    2016-10-01

    Traditionally, trehalose is considered as a protectant to improve the ethanol tolerance of Saccharomyces cerevisiae. In this study, to clarify the changes and roles of trehalose during the bioethanol fermentation, trehalose content and expression of related genes at lag, exponential, and stationary phases (i.e., 2, 8, and 16 h of batch fermentation process) were determined. Although yeast cells at exponential and stationary phase had higher trehalose content than cells at lag phase (P 0.05). Moreover, expression of the trehalose degradation-related genes NTH1 and NTH2 decreased at exponential phase in comparison with that at lag phase; compared with cells at lag phase, cells at stationary phase had higher expression of TPS1, ATH1, NTH1, and NTH2 but lower expression of TPS2. During the lag-exponential phase transition, downregulation of NTH1 and NTH2 promoted accumulation of trehalose, and to some extent, trehalose might confer ethanol tolerance to S. cerevisiae before stationary phase. During the exponential-stationary phase transition, upregulation of TPS1 contributed to accumulation of trehalose, and Tps1 protein might be indispensable in yeast cells to withstand ethanol stress at the stationary phase. Moreover, trehalose would be degraded to supply carbon source at stationary phase.

  5. Feasibility of brewing makgeolli using Pichia anomala Y197-13, a non-Saccharomyces cerevisiae.

    Science.gov (United States)

    Kim, Hye Ryun; Kim, Jae-Ho; Bai, Dong-Hoon; Ahn, ByungHak

    2012-12-01

    Makgeolli is a traditional rice wine favored by the general public in Korea. This study investigated the fermentation and sensory characteristics of using wild yeast strains for brewing makgeolli. A non-Saccharomyces cerevisiae strain was isolated from nuruk and termed Y197-13. It showed 98% similarity to Pichia anomala and had an optimal growth temperature of 25 degrees C. Makgeolli was manufactured using koji, jinju nuruk, and improved nuruk as fermentation agents. Y197-13 makgeolli brewed with koji had alcohol and solids contents of 11.1% and 13.9%, respectively. Sweet sensory characteristics were attributed to residual sugars in makgeolli with 6% alcohol. The makgeolli had a fresh sour taste and carbonated taste. Volatile component analysis showed the isoamyl alcohol, phenylethyl alcohol, isoamyl acetate, and fatty acid, including ethyl oleate and ethyl linoleate, relative peak area was higher in Y197-13 makgeolli than in makgeolli with Saccharomyces cerevisiae. These results suggest the wild yeast, Y197-13, as a candidate for brewing makgeolli.

  6. Industrial systems biology of Saccharomyces cerevisiae enables novel succinic acid cell factory.

    Directory of Open Access Journals (Sweden)

    José Manuel Otero

    demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals.

  7. Physiology of Saccharomyces cerevisiae during cell cycle oscillations.

    Science.gov (United States)

    Duboc, P; Marison, I; von Stockar, U

    1996-10-18

    Synchronized populations of Saccharomyces cerevisiae CBS 426 are characterized by autonomous oscillations of process variables. CO2 evolution rate, O2 uptake rate and heat production rate varied by a factor of 2 for a continuous culture grown at a dilution rate of 0.10 h-1. Elemental analysis showed that the carbon mass fraction of biomass did not change. Since the reactor is not at steady state, the elemental and energy balances were calculated on cumulated quantities, i.e. the integral of the reaction rates. It was possible to show that carbon, degree of reduction and energy balances matched. Application of simple mass balance principles for non-steady state systems indicated that oscillations were basically characterized by changes in biomass production rate. In addition, the amount of intermediates, e.g. ethanol or acetate, produced or consumed was negligible. Growth rate was low during the S-phase (0.075 h-1) and high during the G2, M and G1 phases (0.125 h-1) for a constant dilution rate of 0.10 h-1. However, nitrogen, ash, sulfur and potassium content showed systematic increases during the S-phase (bud initiation). Cell component analyses showed that changes in cellular fractions during oscillations (storage carbohydrate content decreased during the S-phase) were due to changes in production rates, particularly for protein and carbohydrates. Nevertheless, using the data evaluation techniques for dynamic systems presented here, it was shown that storage carbohydrates are not consumed during the S-phase. Only the synthesis rate of the different cell components changed depending on position in cell cycle. The growth process may be divided into two phenomena: the formation of new cells during mitosis with a low yield, and size increase of new born cells with high yield. Both kinetic and stoichiometric coefficients varied with the position in the oscillation: the results showed that biomass structure changed and that specific growth rate, as well as biomass yield

  8. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyes cerevisiae improves ethanol production

    DEFF Research Database (Denmark)

    Roca, Christophe Francois Aime; Nielsen, Jens; Olsson, Lisbeth

    2003-01-01

    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase...

  9. Fps1p channel is the mediator of the major part of glycerol passive diffusion in Saccharomyces cerevisiae : artefacts and re-definitions

    OpenAIRE

    Oliveira, Rui Pedro Soares de; Lages, Fernanda; Graça, Magda Maria Couto da Silva; Lucas, Cândida

    2003-01-01

    Glycerol has been shown to cross Saccharomyces cerevisiae plasma membrane (1) through a H+/symport detected in cells grown on non-fermentable carbon sources, (2) by passive diffusion and (3) through the constitutive Fps1p channel. This has been named a facilitator, for mediating glycerol low affinity transport of the facilitated diffusion type. We present experimental evidence that this kinetics is an artefact created by glycerol kinase activity. Instead, the channel is shown to mediate the m...

  10. Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae

    Science.gov (United States)

    Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert

    2016-01-01

    The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250

  11. Functional profiling of the Saccharomyces cerevisiae genome.

    Science.gov (United States)

    Giaever, Guri; Chu, Angela M; Ni, Li; Connelly, Carla; Riles, Linda; Véronneau, Steeve; Dow, Sally; Lucau-Danila, Ankuta; Anderson, Keith; André, Bruno; Arkin, Adam P; Astromoff, Anna; El-Bakkoury, Mohamed; Bangham, Rhonda; Benito, Rocio; Brachat, Sophie; Campanaro, Stefano; Curtiss, Matt; Davis, Karen; Deutschbauer, Adam; Entian, Karl-Dieter; Flaherty, Patrick; Foury, Francoise; Garfinkel, David J; Gerstein, Mark; Gotte, Deanna; Güldener, Ulrich; Hegemann, Johannes H; Hempel, Svenja; Herman, Zelek; Jaramillo, Daniel F; Kelly, Diane E; Kelly, Steven L; Kötter, Peter; LaBonte, Darlene; Lamb, David C; Lan, Ning; Liang, Hong; Liao, Hong; Liu, Lucy; Luo, Chuanyun; Lussier, Marc; Mao, Rong; Menard, Patrice; Ooi, Siew Loon; Revuelta, Jose L; Roberts, Christopher J; Rose, Matthias; Ross-Macdonald, Petra; Scherens, Bart; Schimmack, Greg; Shafer, Brenda; Shoemaker, Daniel D; Sookhai-Mahadeo, Sharon; Storms, Reginald K; Strathern, Jeffrey N; Valle, Giorgio; Voet, Marleen; Volckaert, Guido; Wang, Ching-yun; Ward, Teresa R; Wilhelmy, Julie; Winzeler, Elizabeth A; Yang, Yonghong; Yen, Grace; Youngman, Elaine; Yu, Kexin; Bussey, Howard; Boeke, Jef D; Snyder, Michael; Philippsen, Peter; Davis, Ronald W; Johnston, Mark

    2002-07-25

    Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

  12. Arsenate and phosphate interaction in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    GENG Chun-nu; ZHU Yong-guan

    2006-01-01

    In the present study, arsenate(As(Ⅴ)) and phosphate(P(Ⅴ)) interactions were investigated in growth, uptake and RNA content in yeast(Saccharomyces cerevisiae). Yeast grew slowly with As(Ⅴ) concentrations increasing in the medium. However, the maximal population density was almost the same among different As(Ⅴ) treatments. It was in the late log phase that yeast growth was augmented by low As(Ⅴ), which was maybe due to the fact that methionine metabolism was stressed by vitamin B6 deprivation, so As(Ⅴ)treatments did not affect maximal population density. However, with P (Ⅴ) concentrations increasing, the maximal population density increased. Therefore, the maximal population density was determined by P (Ⅴ) concentrations in the medium but not by As (Ⅴ)concentrations in the medium. Ycf1p(a tonoplast transpor) transports As(GS)3 into the vacuole, but arsenic(As) remaining in the thalli was 1.27% with As(Ⅴ) exposure for 60 h, from which it can be speculated that the percentage of As transported into vacuole should be lower than 1.27%. However, the percentage of As pumped out of cell was 71.49% with As (Ⅴ) exposure for 68 h. Although two pathways (extrusion and sequestration) were involved in As detoxification in yeast, the extrusion pathway played a major role in As detoxification. RNA content was the highest in the early-log phase and was reduced by As(Ⅴ).

  13. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weinert, Brian T; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chunaram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. Mitochondrial acetylation levels correlated with acetyl-CoA concentration in vivo and acetyl-CoA acetylated lysine residues nonenzymatically in vitro. We developed a method to estimate acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing yeast and is uniformly affected by exposure to acetyl-CoA.

  14. Saccharomyces cerevisiae Is Permissive for Replication of Bovine Papillomavirus Type 1

    OpenAIRE

    Zhao, Kong-Nan; Frazer, Ian H

    2002-01-01

    We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey pa...

  15. Ultrastructural changes of Saccharomyces cerevisiae in response to ethanol stress.

    Science.gov (United States)

    Ma, Manli; Han, Pei; Zhang, Ruimin; Li, Hao

    2013-09-01

    In the fermentative process using Saccharomyces cerevisiae to produce bioethanol, the performance of cells is often compromised by the accumulation of ethanol. However, the mechanism of how S. cerevisiae responds against ethanol stress remains elusive. In the current study, S. cerevisiae cells were cultured in YPD (yeast extract - peptone - dextrose) medium containing various concentrations of ethanol (0%, 2.5%, 5%, 7.5%, 10%, and 15% (v/v)). Compared with the control group without ethanol, the mean cell volume of S. cerevisiae decreased significantly in the presence of 7.5% and 10% ethanol after incubation for 16 h (P < 0.05), and in the presence of 15% ethanol at all 3 sampling time points (1, 8, and 16 h) (P < 0.05). The exposure of S. cerevisiae cells to ethanol also led to an increase in malonyldialdehyde content (P < 0.05) and a decrease in sulfhydryl group content (P < 0.05). Moreover, the observations through transmission electron microscopy enabled us to relate ultrastructural changes elicited by ethanol with the cellular stress physiology. Under ethanol stress, the integrity of the cell membrane was compromised. The swelling or distortion of mitochondria together with the occurrence of a single and large vacuole was correlated with the addition of ethanol. These results suggested that the cell membrane is one of the targets of ethanol, and the degeneration of mitochondria promoted the accumulation of intracellular reactive oxygen species.

  16. Removing cadmium from electroplating wastewater by waste saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    DAI Shu-juan; WEI De-zhou; ZHOU Dong-qin; JIA Chun-yun; WANG Yu-juan; LIU Wen-gang

    2008-01-01

    The appropriate condition and scheme of removing cadmium from electroplating wastewater were investigated by adsorption-precipitation method using waste saccharomyces cerevisiae(WSC) as sorbent. Effect factors on biosorption of cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae and precipitation process of waste saccharomyces cerevisiae after adsorbing cadmium were studied. The results show that removal rate of cadmium is over 88% after 30 min adsorbing under the condition of cadmium concentration 26 mg/L, the dosage of waste saccharomyces cerevisiae 16.25 g/L, temperature 18 ℃, pH 6.0 and precipitation time 4 h. Biosorption-precipitation method is effective to remove cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae. The SEM, infrared spectroscopy and Zeta-potential of the cells show that chemical chelating is the main adsorption form; electrostatic attraction, hydrogen bonding and van der Waals force all function in adsorption process; and ―NH2―,―C=O―,―C=O―NH―,―CH3, ―OH are the main adsorption groups.

  17. Saccharomyces cerevisiae as a starter culture in Mycella.

    Science.gov (United States)

    Hansen, T K; Tempel, T V; Cantor, M D; Jakobsen, M

    2001-09-19

    The potential use of Saccharomyces cerevisiae FB7 as an additional starter culture for the production of Mycella, a Danish Gorgonzola type cheese, was investigated. Two dairy productions of Mycella, each containing batches of experimental cheeses with S. cerevisiae added and reference cheeses without yeast added were carried out. For both experimental and reference cheeses, chemical analysis (pH, a(w), NaCl, water and fat content) were carried out during the ripening period, but no significant differences were found. The evolution of lactic acid bacteria was almost identical in both the experimental and reference cheeses and similar results were found for the number of yeast. S. cerevisiae FB7 was found to be predominant in the core of the experimental cheeses throughout the ripening period, while Debaryomyces hansenii dominated in the reference cheese and on the surface of the experimental cheeses. In the cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of Penicillium roqueforti was observed compared to the reference cheeses. Furthermore, in the experimental cheese, synergistic interactions were also found in the aroma analysis, the degradation of casein and by the sensory analysis. The observed differences indicate a positive contribution to the overall quality of Mycella by S. cerevisiae FB7.

  18. Genetic mapping of quantitative phenotypic traits in Saccharomyces cerevisiae.

    Science.gov (United States)

    Swinnen, Steve; Thevelein, Johan M; Nevoigt, Elke

    2012-03-01

    Saccharomyces cerevisiae has become a favorite production organism in industrial biotechnology presenting new challenges to yeast engineers in terms of introducing advantageous traits such as stress tolerances. Exploring subspecies diversity of S. cerevisiae has identified strains that bear industrially relevant phenotypic traits. Provided that the genetic basis of such phenotypic traits can be identified inverse engineering allows the targeted modification of production strains. Most phenotypic traits of interest in S. cerevisiae strains are quantitative, meaning that they are controlled by multiple genetic loci referred to as quantitative trait loci (QTL). A straightforward approach to identify the genetic basis of quantitative traits is QTL mapping which aims at the allocation of the genetic determinants to regions in the genome. The application of high-density oligonucleotide arrays and whole-genome re-sequencing to detect genetic variations between strains has facilitated the detection of large numbers of molecular markers thus allowing high-resolution QTL mapping over the entire genome. This review focuses on the basic principle and state of the art of QTL mapping in S. cerevisiae. Furthermore we discuss several approaches developed during the last decade that allow down-scaling of the regions identified by QTL mapping to the gene level. We also emphasize the particular challenges of QTL mapping in nonlaboratory strains of S. cerevisiae.

  19. Expression of a codon-optimized β-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose.

    Science.gov (United States)

    Ríos-Fránquez, Francisco Javier; González-Bautista, Enrique; Ponce-Noyola, Teresa; Ramos-Valdivia, Ana Carmela; Poggi-Varaldo, Héctor Mario; García-Mena, Jaime; Martinez, Alfredo

    2017-01-30

    Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L(-1) and 222 IU g(-1), respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L(-1)) as the sole carbon source for 48 h. Ethanol production was 5 g L(-1) after 96 h of culture, which represented a yield of 0.41 g g(-1) of substrate consumed (12 g L(-1)), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

  20. The sequence spectrum of frameshift reversions obtained with a novel adaptive mutation assay in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Erich Heidenreich

    2016-12-01

    Full Text Available Research on the mechanisms of adaptive mutagenesis in resting, i.e. non-replicating cells relies on appropriate mutation assays. Here we provide a novel procedure for the detection of frameshift-reverting mutations in yeast. Proliferation of non-reverted cells in this assay is suppressed by the lack of a fermentable carbon source. The test allele was constructed in a way that the reversions mimic microsatellite instability, a condition often found in cancer cells. We show the cell numbers during these starvation conditions and provide a DNA sequence spectrum of a representative set of revertants. The data in this article support the publication "Glucose starvation as a selective tool for the study of adaptive mutations in Saccharomyces cerevisiae" (Heidenreich and Steinboeck, 2016 [1].

  1. NADP+-dependent glutamate dehydrogenase activity is impaired in mutants of Saccharomyces cerevisiae that lack aconitase.

    Science.gov (United States)

    González, A; Rodríguez, L; Olivera, H; Soberón, M

    1985-10-01

    A mutant of Saccharomyces cerevisiae lacking aconitase did not grow on minimal medium (MM) and had five- to tenfold less NADP+-dependent glutamate dehydrogenase (GDH) activity than the wild-type, although its glutamine synthetase (GS) activity was still inducible. When this mutant was incubated with glutamate as the sole nitrogen source, the 2-oxoglutarate content rose, and the NADP+-dependent GDH activity increased. Furthermore, carbon-limited cultures showed a direct relation between NADP+-dependent GDH activity and the intracellular 2-oxoglutarate content. We propose that the low NADP+-dependent GDH activity found in the mutant was due to the lack of 2-oxoglutarate or some other intermediate of the tricarboxylic acid cycle.

  2. De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Li, Mingji; Kildegaard, Kanchana Rueksomtawin; Chen, Yun;

    2015-01-01

    Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae...... to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.......73±0.05 mg L−1 resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85±0.31 mg L−1 resveratrol from glucose as the sole carbon source. Next we improved...

  3. Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant.

    Science.gov (United States)

    Terrell, S L; Bernard, A; Bailey, R B

    1984-09-01

    An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step allowed us to obtain high biomass concentrations before continuous fermentation. The continuous anaerobic process successfully incorporated a cell-recycle system to optimize the fermentor productivity. Under conditions permitting a low residual sugar concentration (

  4. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  5. Understanding the 3-hydroxypropionic acid tolerance mechanism in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Juncker, Agnieszka; Hallstrom, Bjorn;

    2013-01-01

    a sustainable alternative for production of acrylic acid from renewable feedstocks. We are establishing Saccharomyces cerevisiae as an alternative host for 3HP production. However, 3HP also inhibits yeast grow th at level well below what is desired for commercial applications. Therefore, we are aiming...... to improve 3HP tolerance in S. cerevisiae by applying adaptive evolution approach. We have generated yeast strains with sign ificantly improved capacity for tolerating 3HP when compared to the wild-type. We will present physiolo gical characterization, genome re-sequencing, and transcriptome analysis...

  6. Expression and secretion of Aspergillus niger glucoamylase in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    李文清; 何鸣; 罗进贤

    1995-01-01

    Aspergillus niger glucoamylase GA 1 cDNA was inserted in between the yeast PGK promoter and terminator on plasmid pMA91. The resultant plasmid pMAG69 was introduced into Saccharomyces cerevisiae GRF18 by protoplast transformation. The A niger GA I cDNA was expressed efficiently under the contiol of PGK promoter and 99% of the gene products were secreted into the culture medium using its own signal sequence The recombmant yeast can digest 87% of starch in 2 d in the medium containing 10% starch. The recombinant plasmid pMAG69 can exist stably in 5. cerevisiae.

  7. Genetic mapping of Ty elements in Saccharomyces cerevisiae.

    OpenAIRE

    Klein, H L; Petes, T. D.

    1984-01-01

    We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetical...

  8. Bioethanol production from mixed sugars by Scheffersomyces stipitis free and immobilized cells, and co-cultures with Saccharomyces cerevisiae.

    Science.gov (United States)

    De Bari, Isabella; De Canio, Paola; Cuna, Daniela; Liuzzi, Federico; Capece, Angela; Romano, Patrizia

    2013-09-25

    Bioethanol can be produced from several biomasses including lignocellulosic materials. Besides 6-carbon sugars that represent the prevalent carbohydrates, some of these feedstocks contain significant amounts of 5-carbon sugars. One common limit of the major part of the xylose-fermenting yeasts is the diauxic shift between the uptake of glucose and xylose during the fermentation of mixed syrups. Thus, optimized fermentation strategies are required. In this paper the ability of Scheffersomyces stipitis strain NRRLY-11544 to ferment mixed syrups with a total sugar concentration in the range 40-80 g/L was investigated by using mono cultures, co-cultures with Saccharomyces cerevisiae strain Bakers Yeast Type II and single cultures immobilized in silica-hydrogel films. The experimental design for the fermentations with immobilized cells included the process analysis in function of two parameters: the fraction of the gel in the broth and the concentration of the cells loaded in the gel. Furthermore, for each total sugars level, the fermentative course of S. stipitis was analyzed at several glucose-to xylose ratios. The results indicated that the use of S. stipitis and S. cerevisiae in free co-cultures ensured faster processes than single cultures of S. stipitis either free or immobilized. However, the rapid production of ethanol by S. cerevisiae inhibited S. stipitis and caused a stuck of the process. Immobilization of S. stipitis in silica-hydrogel increased the relative consumption rate of xylose-to-glucose by 2-6 times depending on the composition of the fermentation medium. Furthermore the films performances appeared stable over three weeks of continuous operations. However, on the whole, the final process yields obtained with the immobilized cells were not meaningfully different from that of the free cells. This was probably due to concurrent fermentations operated by the cells released in the broth. Optimization of the carrier characteristics could improve the

  9. Profiling of cytosolic and peroxisomal acetyl-CoA metabolism in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yun Chen

    Full Text Available As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products.

  10. Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.

    Science.gov (United States)

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2015-08-03

    During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation.

  11. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    Science.gov (United States)

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  12. The enantioselective b-keto ester reductions by Saccharomyces cerevisiae

    OpenAIRE

    HASSAN TAJIK; KHALIL TABATABAEIAN; MAHMOOD SHAHBAZI

    2006-01-01

    The enantioselective yeast reduction of aromatic b-keto esters, by use of potassium dihydrogen phosphate, calcium phosphate (monobasic), magnesium sulfate and ammonium tartrate (diammonium salt) (10:1:1:50) in water at pH 7 as a buffer for 72–120 h with 45–90 % conversion to the corresponding aromatic -hydroxy esters was achieved by means of Saccharomyces cerevisiae.

  13. Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

    Science.gov (United States)

    Marques, Wesley Leoricy; Raghavendran, Vijayendran; Stambuk, Boris Ugarte; Gombert, Andreas Karoly

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

  14. Adsorption and Interfacial Electron Transfer of Saccharomyces Cerevisiae

    DEFF Research Database (Denmark)

    Hansen, Allan Glargaard; Boisen, Anja; Nielsen, Jens Ulrik;

    2003-01-01

    We have studied the adsorption and electron-transfer dynamics of Saccharomyces cerevisiae (yeast) iso-l-cytochrome c adsorbed on Au(lll) electrodes in aqueous phosphate buffer media. This cytochrome possesses a thiol group dos e to the protein surface (Cysl02) suitable for linking the protein...

  15. 2μ plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

    Science.gov (United States)

    Strope, Pooja K; Kozmin, Stanislav G; Skelly, Daniel A; Magwene, Paul M; Dietrich, Fred S; McCusker, John H

    2015-12-01

    We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer.

  16. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Vernis, L.; Piskur, Jure; Diffley, J.F.X.

    2003-01-01

    The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well...

  17. The Plasma Membrane of Saccharomyces cerevisiae : Structure, Function, and Biogenesis

    NARCIS (Netherlands)

    VANDERREST, ME; KAMMINGA, AH; NAKANO, A; ANRAKU, Y; POOLMAN, B; KONINGS, WN

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extens

  18. Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Solis Escalante, D.

    2015-01-01

    Glycolysis, a biochemical pathway that oxidizes glucose to pyruvate, is at the core of sugar metabolism in Saccharomyces cerevisiae (bakers’ yeast). Glycolysis is not only a catabolic route involved in energy conservation, but also provides building blocks for anabolism. From an applied perspective,

  19. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

    Science.gov (United States)

    Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

    2016-07-01

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE.

  20. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    Science.gov (United States)

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

  1. (13)C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids.

    Science.gov (United States)

    Ghosh, Amit; Ando, David; Gin, Jennifer; Runguphan, Weerawat; Denby, Charles; Wang, George; Baidoo, Edward E K; Shymansky, Chris; Keasling, Jay D; García Martín, Héctor

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined (13)C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption. These genetic modifications were applied to S. cerevisiae WRY2, a strain that is capable of producing 460 mg/L of free fatty acids. With the addition of ATP citrate lyase and downregulation of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux through the acetyl-CoA production pathway in the cytoplasm. In total, the genetic interventions applied in this work increased fatty acid production by ~70%.

  2. Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in media containing different sugar sources.

    Science.gov (United States)

    Zeng, Wei-Yi; Tang, Yue-Qin; Gou, Min; Xia, Zi-Yuan; Kida, Kenji

    2016-12-01

    Lignocellulosic hydrolysates used for bioethanol production contain a mixture of sugars, with xylose being the second most abundant after glucose. Since xylose is not a natural substrate for Saccharomyces cerevisiae, recombinant S. cerevisiae strongly prefers glucose over xylose, and the fermentation rate and ethanol yield with xylose are both lower than those with glucose. To determine the molecular basis for glucose and xylose fermentation, we used microarrays to investigate the transcriptional difference of a xylose-utilizing industrial strain cultured in both single sugar media and a mixed sugar medium of glucose and xylose. The transcriptomes were nearly identical between glucose metabolizing cells in the glucose alone medium and those in the glucose fermentation phase in the mixed-sugar medium. Whereas the transcriptomes highly differed between the xylose metabolizing cells in the xylose alone medium and those in the xylose fermentation phase in the mixed sugar medium, and the differences mainly involved sulfur metabolism. When the transcriptional profiles were compared between glucose fermentation state and xylose fermentation state, we found the expression patterns of hexose transporters and glucose signaling pathway differed in response to different sugar sources, and the expression levels of the genes involved in gluconeogenesis, the glyoxylate and tricarboxylic acid cycles and respiration increased with xylose, indicating that the xylose-metabolizing cells had high requirements for maintenance energy and lacked the carbon catabolite repression capability. The effect of carbon catabolite repression by glucose lasted after glucose depletion for specific genes to different extents.

  3. Saccharomyces cerevisiae as a model organism: a comparative study.

    Directory of Open Access Journals (Sweden)

    Hiren Karathia

    Full Text Available BACKGROUND: Model organisms are used for research because they provide a framework on which to develop and optimize methods that facilitate and standardize analysis. Such organisms should be representative of the living beings for which they are to serve as proxy. However, in practice, a model organism is often selected ad hoc, and without considering its representativeness, because a systematic and rational method to include this consideration in the selection process is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this work we propose such a method and apply it in a pilot study of strengths and limitations of Saccharomyces cerevisiae as a model organism. The method relies on the functional classification of proteins into different biological pathways and processes and on full proteome comparisons between the putative model organism and other organisms for which we would like to extrapolate results. Here we compare S. cerevisiae to 704 other organisms from various phyla. For each organism, our results identify the pathways and processes for which S. cerevisiae is predicted to be a good model to extrapolate from. We find that animals in general and Homo sapiens in particular are some of the non-fungal organisms for which S. cerevisiae is likely to be a good model in which to study a significant fraction of common biological processes. We validate our approach by correctly predicting which organisms are phenotypically more distant from S. cerevisiae with respect to several different biological processes. CONCLUSIONS/SIGNIFICANCE: The method we propose could be used to choose appropriate substitute model organisms for the study of biological processes in other species that are harder to study. For example, one could identify appropriate models to study either pathologies in humans or specific biological processes in species with a long development time, such as plants.

  4. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  5. 13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of Fatty Acids

    DEFF Research Database (Denmark)

    Ghosh, Amit; Ando, David; Gin, Jennifer

    2016-01-01

    Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined 13C labeling data with comprehensive genome-scale models...... of malate synthase, the engineered strain produced 26% more free fatty acids. Further increases in free fatty acid production of 33% were obtained by knocking out the cytoplasmic glycerol-3-phosphate dehydrogenase, which flux analysis had shown was competing for carbon flux upstream with the carbon flux...... to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source...

  6. Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A

    Directory of Open Access Journals (Sweden)

    Wiebe Marilyn

    2008-07-01

    Full Text Available Abstract Background The yeast Saccharomyces cerevisiae is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of S. cerevisiae but understanding of the oxygen dependence of intracellular flux distributions is still scarce. Results Metabolic flux distributions of S. cerevisiae CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O2 in the inlet gas were quantified by 13C-MFA. Metabolic flux ratios from fractional [U-13C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of S. cerevisiae. While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO2. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway. Conclusion 13C-constrained MFA enabled quantitative determination of

  7. Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

    Directory of Open Access Journals (Sweden)

    Livas Daniela

    2011-08-01

    Full Text Available Abstract Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA, triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in

  8. Pengaruh Variasi Kecepatan Agitasi pada Produksi Β-Glukan dari Saccharomyces cerevisiae

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    Laras Cempaka

    2016-03-01

    Full Text Available β-glucan is very interesting to study because of a variety of benefits that it provides. Saccharomyces cerevisiae is a unicellular yeast which has a β-glucan component of the biggest in the cell wall. This study aimed to describe the effect of agitation speed on the production of β-glucan from S. cerevisiae. Agitation speed plays an important role in cell growth. This research used agitation speed at 80 rpm, 120 rpm and 200 rpm. The research design used was a completely randomized design with three replications. During the fermentation in sixteen hours, several parameters were examined including cell number, pH, glucose and protein of the medium and the crude β-glucan. β-glucan extraction procedures done by adding NaOH 2% solution to the fermented product. Then, the supernatant was neutralized with acetic acid solution. To get the crude deposits of β-glucan, ethanol 96% was added in volume as three times of the supernatant. Production of β-glucan was increas along with the growth of the cell.Data analysis was performed using one way ANOVA test followed by LSD analysis. Production of β-glucan increases with cell growth. pH value, the concentration of carbon source and nitrogen source on the substrate decreased during the fermentation process. β-glucan production also increased as the rising of agitation speed from the 80 rpm until 200 rpm. Rate of β-glucan production in 80 rpm, 120 rpm and 200 rpm were 18.19 μgL-1/ hour, 40.42 μgL-1/ hour, 44.03 μgL-1/ hour, respectively. Based on the experiment results, the most optimum agitation speed for beta-glucan were respectively 200 rpm with beta-glucan content reached 1624.44 µg/L.

  9. Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

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    Sheng, Jiayuan; Stevens, Joseph; Feng, Xueyang

    2016-05-27

    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

  10. Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

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    Basso, Thiago O; de Kok, Stefan; Dario, Marcelo; do Espirito-Santo, Júlio Cézar A; Müller, Gabriela; Schlölg, Paulo S; Silva, Carlos P; Tonso, Aldo; Daran, Jean-Marc; Gombert, Andreas K; van Maris, Antonius J A; Pronk, Jack T; Stambuk, Boris U

    2011-11-01

    Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

  11. Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

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    Sônia C. Melo

    2015-06-01

    Full Text Available Heterologous expression of a putative manganese superoxide dismutase gene (SOD2 of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs located the protein of M. perniciosa (MpSod2p in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

  12. Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae.

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    Leber, Christopher; Polson, Brian; Fernandez-Moya, Ruben; Da Silva, Nancy A

    2015-03-01

    The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes. β-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490mg/L. Combining these two disrupted pathways, a sextuple mutant (Δfaa1 Δfaa4 Δfat1 Δfaa2 Δpxa1 Δpox1) was able to produce 1.3g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1, TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae.

  13. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

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    Míriam Cristina Sakuragui Matuo

    2010-09-01

    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  14. Inhibitory effects of gallic acid ester derivatives on Saccharomyces cerevisiae multidrug resistance protein Pdr5p.

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    Pereira Rangel, Luciana; Fritzen, Márcio; Yunes, Rosendo Augusto; Leal, Paulo César; Creczynski-Pasa, Tânia Beatriz; Ferreira-Pereira, Antônio

    2010-05-01

    Overexpression of the Saccharomyces cerevisiae ABC transporter Pdr5p confers resistance to a range of structurally unrelated xenobiotics. This property allows Pdr5p to be used as a target for novel multidrug resistance reversal reagents or chemosensitizers. Herein, we report the effects of gallic acid derivatives with substitutions either on the ester moiety or in the benzene ring on the activity of Pdr5p. Compounds with a longer side chain (8-16 carbons) resulted in greater inhibition of Pdr5p ATPase. Derivatives with side chains of 8-12 carbons that retained hydroxyl groups on the benzene ring extensively inhibited Pdr5p ATPase activity. These compounds almost completely inhibited the efflux of the Pdr5p fluorescent substrate Rhodamine 6G and at 25 muM chemosensitized the Pdr5p-overexpressing strain AD124567 to fluconazole (0.4 mg mL(-1)). Gallic acid derivatives may be a new class of Pdr5p inhibitors.

  15. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

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    McCammon, M T

    1996-09-01

    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the gl