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.

Kinetics of absorption of carbon dioxide in aqueous amine and carbonate solutions with carbonic anhydrase

NARCIS (Netherlands)

Penders-van Elk, Nathalie J. M. C.; Hamborg, Espen S.; Huttenhuis, Patrick J. G.; Fradette, Sylvie; Carley, Jonathan A.; Versteeg, Geert F.

In the present work the absorption of carbon dioxide in aqueous N-methyldiethanolamine (MDEA) and aqueous sodium carbonate with and without carbonic anhydrase (CA) was studied in a stirred cell contactor in the temperature range 298-333 K. The CA was present as free enzyme and is compared to the

Carborane-based inhibitors of carbonic anhydrases

Czech Academy of Sciences Publication Activity Database

Brynda, Jiří; Pachl, Petr; Šícha, Václav; Fábry, Milan; Grüner, Bohumír; Cígler, Petr; Řezáčová, Pavlína

2015-01-01

Roč. 22, č. 1 (2015), s. 3 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] R&D Projects: GA ČR GA15-05677S Institutional support: RVO:61388963 ; RVO:68378050 ; RVO:61388980 Keywords : carboranes * carbonic anhydrase Subject RIV: CE - Biochemistry

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

DEFF Research Database (Denmark)

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

2004-01-01

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

Crystallization, characterization and preliminary X-ray crystallographic analysis of GK2848, a putative carbonic anhydrase of Geobacillus kaustophilus

International Nuclear Information System (INIS)

Ragunathan, Preethi; Raghunath, Gokul; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

2013-01-01

The expression, purification, characterization and crystallization of GK2848, a carbonic anhydrase from G. kaustophilus, are described. The crystals diffracted to a resolution of 2.70 Å. GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni–NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2 1 2 1 2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur–Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized

Effect of thyroid hormone on the levels of erythrocyte carbonic anhydrase isozymes and 2,3-diphosphoglycerate in rabbits.

Science.gov (United States)

Kondo, T; Taniguchi, N; Ishikawa, N; Ide, H; Takakuwa, E; Murao, M

1978-05-01

Levels of rabbit erythrocyte carbonic anhydrase B and C isozymes were determined in experimental hyperthyroidism using a quantitative immunologic technique. Levels of erythrocyte 2,3-diphosphoglycerate and protein binding iodine were simultaneously determined. Thyroxine and 3,5,3'-triiodothyronine were administered to rabbits orally for 30 days. A significant decrease in carbonic anhydrase B type was observed after 30 days, although no significant change was observed in carbonic anhydrase C type. These findings suggest that the steady state level of carbonic anhydrase B type in red cells is affected by thyroid hormone more readily than that of carbonic anhydrase C type. The level of red cell 2,3-diphosphoglycerate increased markedly after 10 days of treatment, corresponding to the increase of protein binding iodine. The clinical or pathologic significances were discussed in relation to the changes in the levels of these isozymes and 2,3-diphosphglycerate in red cells.

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

International Nuclear Information System (INIS)

Moroney, J.V.; Togasaki, R.K.; Husic, H.D.; Tolbert, N.E.

1987-01-01

Inorganic carbon (C/sub i/) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO 2 . Both air-grown cells, that have a CO 2 concentrating system, and 5% CO 2 -grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C/sub i/) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO 2 -grown cells also accumulated some C/sub i/, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO 2 fixation by high CO 2 -grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO 2 -grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase

In vivo effects of radioactive properties of Tl-201 on human carbonic anhydrase activity

Science.gov (United States)

Sahin, Ali; Senturk, Murat

2017-04-01

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. The most useful radioisotope, Tl-201, decays by electron capture, emitting Hg X-rays ( 70-80 keV), and photons of 135 and 167 keV in 10% total abundance. Therefore, it has good imaging characteristics without excessive patient radiation dose. It is the most popular isotope used for thallium 201 nuclear cardiac stress tests. In the present study, In vivo inhibitory effect of Tl-201 (Thallium-201) on human erythrocyte carbonic anhydrase (CA) activity were investigated.

Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

Czech Academy of Sciences Publication Activity Database

Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

2015-01-01

Roč. 102, SEP 18 (2015), s. 223-232 ISSN 0223-5234 R&D Projects: GA ČR GA15-05677S Grant - others:Fondo di Ateneo per la Ricerca (PRA)(IT) ORME09SPNC Institutional support: RVO:68378050 Keywords : Human carbonic anhydrase * Isoquinoline * Quinoline * X-ray * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.902, year: 2015

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

Directory of Open Access Journals (Sweden)

Joyanta K Modak

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

Ion mobility spectrometry focusing on speciation analysis of metals/metalloids bound to carbonic anhydrase.

Science.gov (United States)

Pessôa, Gustavo de Souza; Pilau, Eduardo Jorge; Gozzo, Fábio Cesar; Arruda, Marco Aurélio Zezzi

2013-09-01

In the present work, traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) was applied to speciation analysis of metalloproteins. The influence of pH on complexation conditions between some metals and bovine carbonic anhydrase was evaluated from pH 6 to 9, as well as the time involved in their complexation (0-24 h). Employing TWIMS-MS, two conformational states of bovine carbonic anhydrase were observed with charge states of +12 and +11; these configurations being evaluated in terms of the folded state of the apo form and this protein (at charge state +11) being linked to barium, lead, copper, and zinc in their divalent forms. Metalloprotein speciation analysis was carried out for copper (Cu(+) and Cu(2+)), lead (Pb(2+) and Pb(4+)), and selenium (Se(4+) and Se(6+)) species complexed with bovine carbonic anhydrase. Mobilities of all complexed species were compared, also considering the apo form of this protein.

SwissProt search result: AK060777 [KOME

Lifescience Database Archive (English)

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

Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish.

Directory of Open Access Journals (Sweden)

Ruben Postel

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

An experimental study on the effect of carbonic anhydrase on the oxygen isotope exchange kinetics and equilibrium in the carbonic acid system

Science.gov (United States)

Uchikawa, J.; Zeebe, R. E.

2011-12-01

Stable oxygen isotopes of marine biogenic carbonates are often depleted in 18O relative to the values expected for thermodynamic equilibrium with ambient seawater. One possibility is that 18O-depletion in carbonates is kinetically controlled. The kinetic isotope effect associated with the hydration of CO2 results in 18O-depleted HCO3-. If the HCO3- is utilized before re-establishing equilibrium with ambient water under rapid calcification, the 18O-depletion will be recorded in carbonates. But one caveat in this kinetic model is the fact that many marine calcifiers posses carbonic anhydrase, a zinc-bearing enzyme that catalyzes the CO2 hydration reaction. It is expected that this enzyme accelerates 18O-equilibration in the carbonic acid system by facilitating direct oxygen isotope exchange between HCO3- and H2O via CO2 hydration. Clearly this argues against the conceptual framework of the kinetic model. Yet the critical variable here is the effectiveness of the carbonic anhydrase, which is likely to depend on its concentration and the carbonate chemistry of the aqueous medium. It is also hitherto unknown whether the presence of carbonic anhydrase alters the equilibrium oxygen isotope fractionations between dissolved carbonate species and water. We performed a series of quantitative inorganic carbonate precipitation experiments to examine the changes in the oxygen isotope equilibration time as a function of carbonic anhydrase concentrations. We conducted experiments at pH 8.3 and 8.9. These pH values are similar to the average surface ocean pH and the elevated pH levels observed within calcification microenvironments of certain corals and planktonic foraminifera. A summary of our new experimental results will be presented.

Carbonic anhydrase (acetazolamide-sensitive esterase) activity in the blood, gill and kidney of the thermally acclimated rainbow trout, Salmo gairdneri

Energy Technology Data Exchange (ETDEWEB)

Houston, A.H.; McCarty, L.S.

1978-04-01

Gill, kidney and blood levels of acetazolamide-sensitive esterase (carbonic anhydrase) activity were estimated at acclimation temperature and at a common temperature (25/sup 0/C) in rainbow trout acclimated to 2, 10, and 18/sup 0/C. Plasma levels of sodium, potassium and chloride were also examined for possible acclimatory variations. Plasma sodium and chloride levels, and the sodium:chloride ratio were unaffected by thermal acclimation; potassium concentrations were significantly elevated at 18/sup 0/C. Significant, but modest changes in renal and branchial carbonic anhydrase activity were observed under physiologically realistic incubation temperature conditions. Blood carbonic anhydrase activity was sharply elevated at higher acclimation temperatures. The data are discussed in relation to the hypothesis that carbonic anhydrase in this relatively stenothermal freshwater salmonid, through its intimate association with the coupled HCO/sub 3//sup -//Cl/sup -/ and H/sup +/ + NH/sub 4//sup +//Na/sup +/ exchange systems may provide for relatively thermostable basal rates of sodium and chloride uptake from the medium and recovery from urine. The renal, and more notably the branchial (Na/sup +//K/sup +/)-simulated ATPase systems, and erythrocytic carbonic anhydrase may then serve primarily as high-temperature amplifiers of sodium and chloride recruitment respectively.

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. IC 50 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.

An Overview of the Bacterial Carbonic Anhydrases

Directory of Open Access Journals (Sweden)

Claudiu T. Supuran

2017-11-01

Full Text Available Bacteria encode carbonic anhydrases (CAs, EC 4.2.1.1 belonging to three different genetic families, the α-, β-, and γ-classes. By equilibrating CO2 and bicarbonate, these metalloenzymes interfere with pH regulation and other crucial physiological processes of these organisms. The detailed investigations of many such enzymes from pathogenic and non-pathogenic bacteria afford the opportunity to design both novel therapeutic agents, as well as biomimetic processes, for example, for CO2 capture. Investigation of bacterial CA inhibitors and activators may be relevant for finding antibiotics with a new mechanism of action.

Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment

Czech Academy of Sciences Publication Activity Database

Závadová, Zuzana; Závada, Jan

2005-01-01

Roč. 13, č. 5 (2005), s. 977-982 ISSN 1021-335X R&D Projects: GA ČR(CZ) GA203/02/0405 Institutional research plan: CEZ:AV0Z50520514 Keywords : carbonic anhydrase IX * cell adhesion * microenvironment Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.572, year: 2005

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

Directory of Open Access Journals (Sweden)

Asahi MG

2015-12-01

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

Radioimmunoassay of rat carbonic anhydrases I and II. Application to central nervous system during ontogenesis

International Nuclear Information System (INIS)

Limozin, Nicole; Filippi, Danielle; Dalmasso, Christiane; Laurent, Georgette

1979-01-01

A specific radioimmunoassay method for rat erythrocyte carbonic anhydrases I and II was developed using a double antibody system. Its sensitivity was in the nanogram range for each of the two isozymes. The method has been applied to the assay of cerebral carbonic anhydrase. Only CAII has been found in brain extracts of perfused rats. Accordingly, the assay of CAI in cerebral tissue can be used to quantify erythrocyte contamination on condition that the ratio CAII/CAI in blood had been worked out. The developmental change in the soluble and the Triton X-100 solubilized brain CAII from birth to adult is reported [fr

Recognition and Binding of a Helix-Loop-Helix Peptide to Carbonic Anhydrase Occurs via Partly Folded Intermediate Structures

Science.gov (United States)

Lignell, Martin; Becker, Hans-Christian

2010-01-01

Abstract We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the

Precipitation of hydrated Mg carbonate with the aid of carbonic anhydrase for CO2 sequestration

Science.gov (United States)

Power, I. M.; Harrison, A. L.; Dipple, G. M.

2011-12-01

Strategies for sequestering CO2 directly from the atmosphere are likely required to achieve the desired reduction in CO2 concentration and avoid the most damaging effects of climate change [1]. Numerous studies have demonstrated the accelerated precipitation of calcium carbonate minerals with the aid of carbonic anhydrase (CA) as a means of sequestering CO2 in solid carbonate form; however, no study has examined precipitation of magnesium carbonate minerals using CA. Precipitation of magnesite (MgCO3) is kinetically inhibited [2]; therefore, Mg2+ must be precipitated as hydrated carbonate minerals. In laboratory experiments, the uptake of atmospheric CO2 into brine solutions (0.1 M Mg) was rate-limiting for the precipitation of dypingite [Mg5(CO3)4(OH)2-5H2O] with initial precipitation requiring 15 days [3]. It was also found that dypingite precipitation outpaced the uptake of CO2 gas into solution. CO2 uptake is limited by the hydration of CO2 to form carbonate ions [4]. Carbonic anhydrase (CA) enzymes are among the fastest known in nature and are able to catalyze the hydration of CO2, i.e., converting CO2(aq) to CO32- and HCO3- [5]. CA plays an important role in the carbon concentrating mechanism of photoautotrophic, chemoautotrophic, and heterotrophic prokaryotes and is involved in pH homeostasis, facilitated diffusion of CO2, ion transport, and the interconversion of CO2 and HCO3- [6]. Introducing CA into buffered Mg-rich solutions should allow for more rapid precipitation of hydrated magnesium carbonate minerals. Batch experiments were conducted using 125 mL flasks containing 100 mL of Millipore deionized water with 0.2 M of MgCl2-6H2O. To buffer pH, 1.0 g of pulverized brucite [Mg(OH)2] or 1.0 g of NaOH was added to the systems, which were amended with Bovine carbonic anhydrase (BCA) (Sigma-Aldrich). Solutions were stirred continuously and kept at room temperature (~22°C) with laboratory air introduced by bubbling. Temperature and pH were measured routinely

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.

Effect of pKa on the kinetics of carbon dioxide absorption in aqueous alkanolamine solutions containing carbonic anhydrase at 298K

NARCIS (Netherlands)

Penders-van Elk, Nathalie J M C; Fradette, Sylvie; Versteeg, Geert F.

2015-01-01

The absorption of carbon dioxide in various aqueous alkanolamine solutions have been studied with and without carbonic anhydrase respectively in a stirred cell reactor at 298K. The examined alkanolamines were: N,N-diethylethanolamine (DEMEA), N,N-dimethylethanolamine (DMMEA), monoethanolamine (MEA),

CO2 Mass transfer model for carbonic anhydrase-enhanced aqueous MDEA solutions

DEFF Research Database (Denmark)

Gladis, Arne Berthold; Deslauriers, Maria Gundersen; Neerup, Randi

2018-01-01

In this study a CO2 mass transfer model was developed for carbonic anhydrase-enhanced MDEA solutions based on a mechanistic kinetic enzyme model. Four different enzyme models were compared in their ability to predict the liquid side mass transfer coefficient at temperatures in the range of 298...

Combined Effect of Temperature and pKa on the Kinetics of Absorption of Carbon Dioxide in Aqueous Alkanolamine and Carbonate Solutions with Carbonic Anhydrase

NARCIS (Netherlands)

Penders-Van Elk, Nathalie J M C; Oversteegen, S. Martijn; Versteeg, Geert F.

2016-01-01

In present work the absorption of carbon dioxide in aqueous N-methyldiethanolamine, N,N-dimethylethanolamine, and triisopropanolamine solutions with and without the enzyme carbonic anhydrase has been studied in a stirred cell reactor at temperatures varying between 278 and 313 K, at an alkanolamine

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.

Gastric Hyperplasia in Mice With Targeted Disruption of the Carbonic Anhydrase Gene Car9

Czech Academy of Sciences Publication Activity Database

Ortova Gut, M.; Parkkila, S.; Vernerová, Z.; Rohde, E.; Závada, Jan; Höcker, M.; Pastorek, J.; Karttunen, T.; Gibadulinová, G.; Závadová, Zuzana; Knobeloch, K. P.; Wiedenmann, B.; Svoboda, Jan; Horak, I.; Pastoreková, S.

2002-01-01

Roč. 123, č. 12 (2002), s. 1889-1903 ISSN 0016-5085 R&D Projects: GA ČR GV312/96/K205 Keywords : Carbonic Anhydrases * Knock-aou * Differantiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.440, year: 2002

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.

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

International Nuclear Information System (INIS)

Weiwei, Zhang; Hu, Renming

2009-01-01

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.

The effect of L-carnitine on carbonic anhydrase level in rats exposed ...

African Journals Online (AJOL)

STORAGESEVER

2009-07-06

Jul 6, 2009 ... effects of dantrolene on carbonic anhydrase enzyme activities. Biol. Pharm. Bull. 27: 613-616. Gülçin I, Küfrevioğlu Öİ, Oktay M (2005). Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibition effects of some chemicals on the enzyme activity. J. Enzym. Inhib. Med.

Carbonic Anhydrase: An Efficient Enzyme with Possible Global Implications

Directory of Open Access Journals (Sweden)

Christopher D. Boone

2013-01-01

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

Gastric hyperplasia in mice with targeted disruption of the carbonic anhydrase gene Car9

Czech Academy of Sciences Publication Activity Database

Ortova-Gut, M.; Parkkila, S.; Vernerová, Z.; Rohde, E.; Závada, Jan; Hocker, M.; Pastorek, J.; Karttunen, T.; Gibadulinová, A.; Závadová, Zuzana; Knobeloch, K.-P.; Wiedernmann, B.; Svoboda, Jan; Horak, I.; Pastoreková, S.

2002-01-01

Roč. 123, č. 6 (2002), s. 1889-1903 ISSN 0016-5085 R&D Projects: GA ČR GV312/96/K205 Institutional research plan: CEZ:AV0Z5052915 Keywords : mouse carbonic anhydrase Car9 * gastric hyperplasia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 13.440, year: 2002

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

Science.gov (United States)

Yao, Zongli; Guo, Wenfei; Lai, Qifang; Shi, Jianquan; Zhou, Kai; Qi, Hongfang; Lin, Tingting; Li, Ziniu; Wang, Hui

2016-01-01

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.

β-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis.

Directory of Open Access Journals (Sweden)

Laura Medina-Puche

Full Text Available The plant hormone salicylic acid (SA is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1 and NON RECOGNITION OF BTH-4 (NRB4 are required for the response to SA in Arabidopsis (Arabidopsis thaliana. Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, βCA1 and βCA2, are β-carbonic anhydrase family proteins. Since double mutant βca1 βca2 plants did not show any obvious phenotype, we investigated other βCAs and found that NRB4 also interacts with βCA3 and βCA4. Moreover, several βCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between βCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4-βCA1-NPR1. The quintuple mutant βca1 βca2 βca3 βca4 βca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.

Electron Spin Resonance Studies of Carbonic Anhydrase: Transition Metal Ions and Spin-Labeled Sulfonamides*

Science.gov (United States)

Taylor, June S.; Mushak, Paul; Coleman, Joseph E.

1970-01-01

Electron spin resonance (esr) spectra of Cu(II) and Co(II) carbonic anhydrase, and a spin-labeled sulfonamide complex of the Zn(II) enzyme, are reported. The coordination geometry of Cu(II) bound in the enzyme appears to have approximately axial symmetry. Esr spectra of enzyme complexes with metal-binding anions also show axial symmetry and greater covalency, in the order ethoxzolamide cyanide complex suggests the presence of two, and probably three, equivalent nitrogen ligands from the protein. Esr spectra of the Co(II) enzyme and its complexes show two types of Co(II) environment, one typical of the native enzyme and the 1:1 CN- complex, and one typical of a 2:1 CN- complex. Co(II) in the 2:1 complex appears to be low-spin and probably has a coordination number of 5. Binding of a spin-labeled sulfonamide to the active center immobilizes the free radical. The similarity of the esr spectra of spin-labeled Zn(II) and Co(II) carbonic anhydrases suggests that the conformation at the active center is similar in the two metal derivatives. PMID:4320976

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.

Hydroxylamine-O-sulfonamide is a versatile lead compound for the development of carbonic anhydrase inhibitors.

Science.gov (United States)

Di Fiore, Anna; Vergara, Alessandro; Caterino, Marco; Alterio, Vincenzo; Monti, Simona M; Ombouma, Joanna; Dumy, Pascal; Vullo, Daniela; Supuran, Claudiu T; Winum, Jean-Yves; De Simone, Giuseppina

2015-07-21

Hydroxylamine-O-sulfonamide, a molecule incorporating two zinc-binding groups (ZBGs), has been investigated as a carbonic anhydrase inhibitor (CAI) by means of kinetic, crystallographic and Raman spectroscopy studies, highlighting interesting results on its mechanism of action. These data can be exploited to design new, effective and selective CAIs.

Radioimmunoassay of human muscle carbonic anhydrase III in dystrophic states

Energy Technology Data Exchange (ETDEWEB)

Heath, R.; Jeffery, S.; Carter, N. (Department of Child Health, St. George' s Hospital Medical School, London (UK))

1982-03-12

A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4..mu..g/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease.

Radioimmunoassay of human muscle carbonic anhydrase III in dystrophic states

International Nuclear Information System (INIS)

Heath, R.; Jeffery, S.; Carter, N.

1982-01-01

A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4μg/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease. (Auth.)

Comparison of the Kinetic Promoters Piperazine and Carbonic Anhydrase for CO₂ Absorption

DEFF Research Database (Denmark)

Gladis, Arne; Gundersen, Maria T.; Thomsen, Kaj

2017-01-01

Kinetic promoter that enhance the reaction kinetics with CO2 are enabling the use of the low heat of reaction of slow absorbing solvents like MDEA. Mass transfer experiments with 30 wt% MDEA promoted by either by 1.7 and 8.5g/L enzyme carbonic anhydrase (CA) or 5 wt% piperazine (PZ) where conduct...

Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II

Energy Technology Data Exchange (ETDEWEB)

Budayova-Spano, Monika [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble (France); Fisher, S. Zoë [Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States); Dauvergne, Marie-Thérèse [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Agbandje-McKenna, Mavis [Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States); Silverman, David N. [Department of Pharmacology and Therapeutics, PO Box 100267, University of Florida, Gainesville, FL 32610 (United States); Myles, Dean A. A. [European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France); Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [Department of Biochemistry and Molecular Biology, PO Box 100245, University of Florida, Gainesville, FL 32610 (United States); European Molecular Biology Laboratory Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble (France)

2006-01-01

This article reports the production, crystallization and X-ray structure determination of perdeuterated human carbonic anhydrase (HCA II). The refined structure is shown to be highly isomorphous with hydrogenated HCA II, especially with regard to the active site architecture and solvent network. Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H{sub 2}O/OH{sup −}) and the proton-shuttling residue His64. This distance (∼7.5 Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (∼1.0 Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.

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...

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.

Carbon- versus sulphur-based zinc binding groups for carbonic anhydrase inhibitors?

Science.gov (United States)

Supuran, Claudiu T

2018-12-01

A set of compounds incorporating carbon-based zinc-binding groups (ZBGs), of the type PhX (X = COOH, CONH 2 , CONHNH 2 , CONHOH, CONHOMe), and the corresponding derivatives with sulphur(VI)-based ZBGs (X = SO 3 H, SO 2 NH 2 , SO 2 NHNH 2 , SO 2 NHOH, SO 2 NHOMe) were tested as inhibitors of all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1), CA I-XV. Three factors connected with the ZBG influenced the efficacy as CA inhibitor (CAI) of the investigated compounds: (i) the pKa of the ZBG; (ii) its geometry (tetrahedral, i.e. sulphur-based, versus trigonal, i.e. carbon-based ZBGs), and (iii) orientation of the organic scaffold induced by the nature of the ZBG. Benzenesulphonamide was the best inhibitor of all isoforms, but other ZBGs led to interesting inhibition profiles, although with an efficacy generally reduced when compared to the sulphonamide. The nature of the ZBG also influenced the CA inhibition mechanism. Most of these derivatives were zinc binders, but some of them (sulfonates, carboxylates) may interact with the enzyme by anchoring to the zinc-coordinated water molecule or by other inhibition mechanisms (occlusion of the active site entrance, out of the active site binding, etc.). Exploring structurally diverse ZBGs may lead to interesting new developments in the field of CAIs.

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients

Czech Academy of Sciences Publication Activity Database

Závada, Jan; Závadová, Zuzana; Zaťovičová, M.; Hyršl, L.; Kawaciuk, I.

2003-01-01

Roč. 89, - (2003), s. 1067-1071 ISSN 0007-0920 R&D Projects: GA ČR GA301/99/0356 Institutional research plan: CEZ:AV0Z5052915 Keywords : carbonic anhydrase IX * tumor antigens * cancer diagnostics Subject RIV: EC - Immunology Impact factor: 3.894, year: 2003

Crucial role of carbonic anhydrase IX in tumorigenicity of xenotransplanted adult T-cell leukemia-derived cells.

Science.gov (United States)

Nasu, Kentaro; Yamaguchi, Kazunori; Takanashi, Tomoka; Tamai, Keiichi; Sato, Ikuro; Ine, Shoji; Sasaki, Osamu; Satoh, Kennichi; Tanaka, Nobuyuki; Tanaka, Yuetsu; Fukushima, Takuya; Harigae, Hideo; Sugamura, Kazuo

2017-03-01

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates cellular pH, is upregulated in various solid tumors, and is considered to be a therapeutic target. Here, we describe the essential role of CA9 in the tumorigenicity of cells derived from human adult T-cell leukemia/lymphoma (ATL). We previously established the highly tumorigenic ST1-N6 subline from the ATL-derived ST1 cell line by serial xenotransplantation in NOG mice. In the present study, we first show that CA9 expression is strongly enhanced in ST1-N6 cells. We then sorted ST1 cells by high or low CA9 expression and established ST1-CA9 high and ST1-CA9 low sublines. ST1-CA9 high cells, like ST1-N6 cells, were more strongly tumorigenic than ST1-CA9 low or parental ST1 cells when injected into NOG mice. Knockdown of CA9 with shRNAs suppressed the ability of ST1-CA9 high cells to initiate tumors, and the tumorigenicity of ST1 cells was significantly enhanced by introducing wild-type CA9 or a CA9 mutant with deletion of an intracytoplasmic domain. However, a CA9 with point mutations in the catalytic site did not increase the tumorigenicity of ST1 cells. Furthermore, we detected a small population of CA9 + CD25 + cells in lymph nodes of ATL patients. These findings suggest that CA9, and particularly its carbonic anhydrase activity, promotes the tumorigenicity of ATL-derived cells and may be involved in malignant development of lymphoma-type ATL. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Carbon dioxide fixation in isolated Kalanchoe chloroplasts

Energy Technology Data Exchange (ETDEWEB)

Levi, C.; Gibbs, M.

1975-07-01

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 ..mu..moles of CO/sub 2/ per milligram of chlorophyll per hour. The dark rate of fixation was about 1 percent of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1, 6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO/sub 2/ fixation were primarily those of the photosynthetic carbon reduction cycle. (auth)

Intramolecular oxidative deselenization of acylselenoureas: a facile synthesis of benzoxazole amides and carbonic anhydrase inhibitors.

Science.gov (United States)

Angeli, A; Peat, T S; Bartolucci, G; Nocentini, A; Supuran, C T; Carta, F

2016-12-28

A mild, efficient and one pot procedure to access benzoxazoles using easily accessible acylselenoureas as starting materials has been discovered. Mechanistic studies revealed a pH dependent intramolecular oxidative deselenization, with ring closure due to an intramolecular nucleophilic attack of a phenoxide ion. All the benzoxazoles herein reported possessed a primary sulfonamide zinc binding group and showed effective inhibitory action on the enzymes, carbonic anhydrases.

Hydrogen/deuterium fractionation factors of the aqueous ligand of cobalt in Co(H2O)62+ and Co(II)-substituted carbonic anhydrase

International Nuclear Information System (INIS)

Kassebaum, J.W.

1988-01-01

The author has measured the hydrogen/deuterium fractionation factor for the rapidly exchanging aqueous ligands of cobalt in Co(H 2 O) 6 2+ and in three Co(II)-substituted isozymes of carbonic anhydrase. The fractionation factor was determined from NMR relaxation rates at 300 MHz of the protons of water in mixed solutions of H 2 O and D 2 O containing these complexes. In each case, the paramagnetic contribution to 1/T 2 was greater than to 1/T 1 , consistent with a chemical shift mechanism affecting 1/T 2 . The fractionation factors obtained from T 2 were 0.73 ± 0.02 for Co(H 2 O) 6 2+ , 0.72 ± 0.02 for Co(II)-substituted carbonic anhydrase I, 0.77 ± 0.01 for Co(II)-substituted carbonic anhydrase II, and 1.00 ± 0.07 for Co(Il)-substituted carbonic anhydrase III. He concluded that fractionation factors in these cases determined from T 1 and T 2 measured isotope preferences for different populations of ligand sites. Since T 2 has a large contribution from a chemical shift mechanism, the fractionation factor determined from T 2 has a large contribution of the fractionation of inner shell ligands. The fractionation factor of Co(H 2 O) 6 2+ was used to interpret the solvent hydrogen isotope effects on the formation of complexes of cobalt with the bidentate ligands glycine, N,N-dimethylglycine, and acetylacetone. The contribution of the fractionation factor of the inner water shell in Co(H 2 O) 6 2+ did not account completely for the measured isotope effect, and that the hydrogen/deuterium fractionation of outer shell water makes a large contribution to the isotope effect on the formation of these complexes

Synthesis and discovery of potent carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase enzymes inhibitors: The novel N,N'-bis-cyanomethylamine and alkoxymethylamine derivatives.

Science.gov (United States)

Taslimi, Parham; Caglayan, Cuneyt; Farzaliyev, Vagif; Nabiyev, Oruj; Sujayev, Afsun; Turkan, Fikret; Kaya, Ruya; Gulçin, İlhami

2018-04-01

During this investigation, N,N'-bis-azidomethylamines, N,N'-bis-cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N-bis-cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α-glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K i values in the range of 0.15-13.31 nM for α-glycosidase, 2.77-15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12-21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33-73.23 nM for AChE, and 3.84-48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances. © 2018 Wiley Periodicals, Inc.

Acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates CO2 removal from blood

OpenAIRE

Arazawa, D. T.; Kimmel, J. D.; Finn, M.C.; Federspiel, W. J.

2015-01-01

The use of extracorporeal carbon dioxide removal (ECCO2R) is well established as a therapy for patients suffering from acute respiratory failure. Development of next generation low blood flow (< 500 mL/min) ECCO2R devices necessitates more efficient gas exchange devices. Since over 90% of blood CO2 is transported as bicarbonate (HCO3−), we previously reported development of a carbonic anhydrase (CA) immobilized bioactive hollow fiber membrane (HFM) which significantly accelerates CO2 removal ...

Molecular Cloning and Characterization of Carbonic Anhydrase XII from Pufferfish (Takifugu rubripes

Directory of Open Access Journals (Sweden)

Kanij Rukshana Sumi

2018-03-01

Full Text Available In this study, an 1888-bp carbonic anhydrase XII (CA XII sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68–56% identity with the large yellow croaker (Larimichthys crocea, tilapia (Oreochromis niloticus, and Asian arowana (Scleropages formosus CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358, two of which (Cys-21 and Cys-201 could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT-PCR, quantitative PCR (q-PCR, and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.

Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplasts 1

Science.gov (United States)

Levi, Carolyn; Gibbs, Martin

1975-01-01

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO2 per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO2 fixation were primarily those of the photosynthetic carbon reduction cycle. PMID:16659249

Bioinformatics Approach Based Research of Profile Protein Carbonic Anhydrase II Analysis as a Potential Candidate Cause Autism for The Variation of Learning Subjects Biotechnology

Directory of Open Access Journals (Sweden)

Dian Eka A. F. Ningrum

2017-03-01

Full Text Available This study aims to determine the needs of learning variations on Biotechnology courses using bioinformatics approaches. One example of applied use of bioinformatics in biotechnology course is the analysis of protein profiles carbonic anhydrase II as a potential cause of autism candidate. This research is a qualitative descriptive study consisted of two phases. The first phase of the data obtained from observations of learning, student questionnaires, and questionnaires lecturer. Results from the first phase, namely the need for variations learning in Biotechnology course using bioinformatics. Collecting data on the second stage uses three webserver to predict the target protein and scientific articles. Visualization of proteins using PyMOL software. 3 based webserver which is used, the candidate of target proteins associated with autism is carbonic anhydrase II. The survey results revealed that the protein carbonic anhydrase II as a potential candidate for the cause of autism classified metaloenzim are able to bind with heavy metals. The content of heavy metals in autistic patients high that affect metabolism. This prediction of protein candidate cause autism is applied use to solve the problem in society, so that can achieve the learning outcome in biotechnology course.

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.

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

Science.gov (United States)

Krungkrai, Sudaratana R; Krungkrai, Jerapan

2011-01-01

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 Zn2+-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. PMID:23569766

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells.

Science.gov (United States)

Ghandour, M S; Parkkila, A K; Parkkila, S; Waheed, A; Sly, W S

2000-11-01

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

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.

Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives

Directory of Open Access Journals (Sweden)

Himadri Bose

2017-08-01

Full Text Available All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives.

Science.gov (United States)

Bose, Himadri; Satyanarayana, Tulasi

2017-01-01

All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA) is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO 3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

In folio study of carbonic anhydrase and Rubisco activities in higher C3 plants using 18O and mass spectrometry

International Nuclear Information System (INIS)

Peltier, G.; Despax, V.; Dimon, B.; Rumeau, D.; Tourneux, C.

1994-01-01

This document studies the effects of a mild water stress and carbonic anhydrase activity by ethoxyzolamide (EZA) on the diffusion of CO 2 in leaves, by 18 O labelling of O 2 and of CO 2 associated to mass spectrometry. (A.B.). 5 refs., 2 figs

Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis.

Science.gov (United States)

Furla, P; Allemand, D; Orsenigo, M N

2000-04-01

Symbiotic cnidarians absorb inorganic carbon from seawater to supply intracellular dinoflagellates with CO(2) for their photosynthesis. To determine the mechanism of inorganic carbon transport by animal cells, we used plasma membrane vesicles prepared from ectodermal cells isolated from tentacles of the sea anemone, Anemonia viridis. H(14)CO(-)(3) uptake in the presence of an outward NaCl gradient or inward H(+) gradient, showed no evidence for a Cl(-)- or H(+)- driven HCO(-)(3) transport. H(14)CO(-)(3) and (36)Cl(-) uptakes were stimulated by a positive inside-membrane diffusion potential, suggesting the presence of HCO(-)(3) and Cl(-) conductances. A carbonic anhydrase (CA) activity was measured on plasma membrane (4%) and in the cytoplasm of the ectodermal cells (96%) and was sensitive to acetazolamide (IC(50) = 20 nM) and ethoxyzolamide (IC(50) = 2.5 nM). A strong DIDS-sensitive H(+)-ATPase activity was observed (IC(50) = 14 microM). This activity was also highly sensitive to vanadate and allyl isothiocyanate, two inhibitors of P-type H(+)-ATPases. Present data suggest that HCO(-)(3) absorption by ectodermal cells is carried out by H(+) secretion by H(+)-ATPase, resulting in the formation of carbonic acid in the surrounding seawater, which is quickly dehydrated into CO(2) by a membrane-bound CA. CO(2) then diffuses passively into the cell where it is hydrated in HCO(-)(3) by a cytosolic CA.

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

Science.gov (United States)

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3 −) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions. PMID:19365544

Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Directory of Open Access Journals (Sweden)

Skander Elleuche

Full Text Available Carbon dioxide (CO(2 is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3 (- reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs. These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3 as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of

Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Science.gov (United States)

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and

Construction of a system for heterologous production of carbonic anhydrase from Plasmodium falciparum in Pichia pastoris

OpenAIRE

Gullberg, Erik

2008-01-01

Malaria is one of the biggest current global health problems, and with the increasing occurance of drug resistant Plasmodium falciparum strains, there is an urgent need for new antimalarial drugs. Given the important role of carbonic anhydrase in Plasmodium falciparum (PfCA), it is a potential novel drug target. Heterologous expression of malaria proteins is problematic due to the unusual codon usage of the Plasmodium genome, so to overcome this problem a synthetic PfCA gene was designed, opt...

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.

Spectroscopic characterization of furosemide binding to human carbonic anhydrase II.

Science.gov (United States)

Ranjbar, Samira; Ghobadi, Sirous; Khodarahmi, Reza; Nemati, Houshang

2012-05-01

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of Förster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Growth and Extracellular Carbonic Anhydrase Activity of Zooxanthellae Symbiodinium sp. in Response of Zinc Enrichment

Directory of Open Access Journals (Sweden)

WIDIASTUTI KARIM

2011-12-01

Full Text Available Coral reef communities contain a wide variety of mutualistic associations none more important than the relationship between corals and their symbiotic dinoflagellates of the genus Symbiodinium sp., commonly referred to as zooxanthellae. The function of Zinc (Zn as cofactor of several enzyme systems such as extracellular carbonic anhydrase (extracellular CA which catalyzes the interconversion of HCO3- and CO2. Concentrations of dissolved Zn in oligothropic waters are often very low therefore may limit the growth of zooxanthellae and their ability to fix CO2 from seawater via the carbonic anhydrase. The aim of this research is to investigate the effect of various concentrations of Zn on the growth and extracellular CA activity in zooxanthellae. Cell density was monitored daily by enumeration with hemocytometer-type chamber (0.1 mm. Extracellular CA was measured in homogenized intact whole cell by a pH drift assay. Results revealed that Zn status strongly influences the growth rate and extracelullar CA activity in zooxanthellae. The specific growth rate and cell density increased two-fold whilst extracelullar CA activity increased 10.5 times higher than that in control with increasing concentrations of Zn from 0 to 80 nM, but decreased when Zn was over 80 nM. Under a concentration of 80 nM was not Zn limited culture, consequently the growth rate of zooxanthellae not dependent on CO2 concentration yet offset by extracelullar CA activity.

Growth and Extracellular Carbonic Anhydrase Activity of Zooxanthellae Symbiodinium sp. in Response of Zinc Enrichment

Directory of Open Access Journals (Sweden)

WIDIASTUTI KARIM

2011-12-01

Full Text Available Coral reef communities contain a wide variety of mutualistic associations none more important than the relationship between corals and their symbiotic dinoflagellates of the genus Symbiodinium sp., commonly referred to as zooxanthellae. The function of Zinc (Zn as cofactor of several enzyme systems such as extracellular carbonic anhydrase (extracellular CA which catalyzes the interconversion of HCO3− and CO2. Concentrations of dissolved Zn in oligothropic waters are often very low therefore may limit the growth of zooxanthellae and their ability to fix CO2 from seawater via the carbonic anhydrase. The aim of this research is to investigate the effect of various concentrations of Zn on the growth and extracellular CA activity in zooxanthellae. Cell density was monitored daily by enumeration with hemocytometer-type chamber (0.1 mm. Extracellular CA was measured in homogenized intact whole cell by a pH drift assay. Results revealed that Zn status strongly influences the growth rate and extracelullar CA activity in zooxanthellae. The specific growth rate and cell density increased two-fold whilst extracelullar CA activity increased 10.5 times higher than that in control with increasing concentrations of Zn from 0 to 80 nM, but decreased when Zn was over 80 nM. Under a concentration of 80 nM was not Zn limited culture, consequently the growth rate of zooxanthellae not dependent on CO2 concentration yet offset by extracelullar CA activity.

Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

International Nuclear Information System (INIS)

Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

2013-01-01

Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site

Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

Energy Technology Data Exchange (ETDEWEB)

Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610 (United States)

2013-05-01

Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

Carbon-concentrating mechanisms in seagrasses.

Science.gov (United States)

Larkum, Anthony William D; Davey, Peter A; Kuo, John; Ralph, Peter J; Raven, John A

2017-06-01

Seagrasses are unique angiosperms that carry out growth and reproduction submerged in seawater. They occur in at least three families of the Alismatales. All have chloroplasts mainly in the cells of the epidermis. Living in seawater, the supply of inorganic carbon (Ci) to the chloroplasts is diffusion limited, especially under unstirred conditions. Therefore, the supply of CO2 and bicarbonate across the diffusive boundary layer on the outer side of the epidermis is often a limiting factor. Here we discuss the evidence for mechanisms that enhance the uptake of Ci into the epidermal cells. Since bicarbonate is plentiful in seawater, a bicarbonate pump might be expected; however, the evidence for such a pump is not strongly supported. There is evidence for a carbonic anhydrase outside the outer plasmalemma. This, together with evidence for an outward proton pump, suggests the possibility that local acidification leads to enhanced concentrations of CO2 adjacent to the outer tangential epidermal walls, which enhances the uptake of CO2, and this could be followed by a carbon-concentrating mechanism (CCM) in the cytoplasm and/or chloroplasts. The lines of evidence for such an epidermal CCM are discussed, including evidence for special 'transfer cells' in some but not all seagrass leaves in the tangential inner walls of the epidermal cells. It is concluded that seagrasses have a CCM but that the case for concentration of CO2 at the site of Rubisco carboxylation is not proven. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Effect of carbonic anhydrase on silicate weathering and carbonate formation at present day CO₂ concentrations compared to primordial values.

Science.gov (United States)

Xiao, Leilei; Lian, Bin; Hao, Jianchao; Liu, Congqiang; Wang, Shijie

2015-01-13

It is widely recognized that carbonic anhydrase (CA) participates in silicate weathering and carbonate formation. Nevertheless, it is still not known if the magnitude of the effect produced by CA on surface rock evolution changes or not. In this work, CA gene expression from Bacillus mucilaginosus and the effects of recombination protein on wollastonite dissolution and carbonate formation under different conditions are explored. Real-time fluorescent quantitative PCR was used to explore the correlation between CA gene expression and sufficiency or deficiency in calcium and CO₂ concentration. The results show that the expression of CA genes is negatively correlated with both CO₂ concentration and ease of obtaining soluble calcium. A pure form of the protein of interest (CA) is obtained by cloning, heterologous expression, and purification. The results from tests of the recombination protein on wollastonite dissolution and carbonate formation at different levels of CO₂ concentration show that the magnitudes of the effects of CA and CO₂ concentration are negatively correlated. These results suggest that the effects of microbial CA in relation to silicate weathering and carbonate formation may have increased importance at the modern atmospheric CO₂ concentration compared to 3 billion years ago.

Cancer Drug Development of Carbonic Anhydrase Inhibitors beyond the Active Site

Directory of Open Access Journals (Sweden)

Srishti Singh

2018-04-01

Full Text Available Carbonic anhydrases (CAs catalyze the reversible hydration of carbon dioxide to produce bicarbonate and a proton. Multiple CA isoforms are implicated in a range of diseases, including cancer. In solid tumors, continuously dividing cells create hypoxic conditions that eventually lead to an acidic microenvironment. Hypoxic tumor cells have different mechanisms in place to regulate and adjust the surrounding microenvironment for survival. These mechanisms include expression of CA isoform IX (CA IX and XII (CA XII. These enzymes help maintain a physiological intracellular pH while simultaneously contributing to an acidic extracellular pH, leading to tumor cell survival. Expression of CA IX and CA XII has also been shown to promote tumor cell invasion and metastasis. This review discusses the characteristics of CA IX and CA XII, their mechanism of action, and validates their prospective use as anticancer targets. We discuss the current status of small inhibitors that target these isoforms, both classical and non-classical, and their future design in order to obtain isoform-specificity for CA IX and CA XII. Biologics, such as monoclonal antibodies, monoclonal-radionuclide conjugated chimeric antibodies, and antibody-small molecule conjugates are also discussed.

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.

Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

Science.gov (United States)

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

2016-01-01

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.

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.

In folio study of carbonic anhydrase and Rubisco activities in higher C{sub 3} plants using {sup 18}O and mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Peltier, G.; Despax, V.; Dimon, B.; Rumeau, D.; Tourneux, C.

1994-12-31

This document studies the effects of a mild water stress and carbonic anhydrase activity by ethoxyzolamide (EZA) on the diffusion of CO{sub 2} in leaves, by {sup 18}O labelling of O{sub 2} and of CO{sub 2} associated to mass spectrometry. (A.B.). 5 refs., 2 figs.

Mimic Carbonic Anhydrase Using Metal-Organic Frameworks for CO2 Capture and Conversion.

Science.gov (United States)

Jin, Chaonan; Zhang, Sainan; Zhang, Zhenjie; Chen, Yao

2018-02-19

Carbonic anhydrase (CA) is a zinc-containing metalloprotein, in which the Zn active center plays the key role to transform CO 2 into carbonate. Inspired by nature, herein we used metal-organic frameworks (MOFs) to mimic CA for CO 2 conversion, on the basis of the structural similarity between the Zn coordination in MOFs and CA active center. The biomimetic activity of MOFs was investigated by detecting the hydrolysis of para-nitrophenyl acetate, which is a model reaction used to evaluate CA activity. The biomimetic materials (e.g., CFA-1) showed good catalytic activity, and excellent reusability, and solvent and thermal stability, which is very important for practical applications. In addition, ZIF-100 and CFA-1 were used to mimic CA to convert CO 2 gas, and exhibited good efficiency on CO 2 conversion compared with those of other porous materials (e.g., MCM-41, active carbon). This biomimetic study revealed a novel CO 2 treatment method. Instead of simply using MOFs to absorb CO 2 , ZIF-100 and CFA-1 were used to mimic CA for in situ CO 2 conversion, which provides a new prospect in the biological and industrial applications of MOFs.

Overlap of epitopes recognized by anti-carbonic anhydrase I IgG in patients with malignancy-related aplastic anemia-like syndrome and in patients with aplastic anemia

Czech Academy of Sciences Publication Activity Database

Jankovičová, B.; Škultéty, L'udovít; Dubrovčáková, M.; Stern, M.; Bílková, Z.; Lakota, J.

2013-01-01

Roč. 153, 1-2 (2013), s. 47-49 ISSN 0165-2478 Institutional support: RVO:61388971 Keywords : Carbonic anhydrase I * Epitope extraction * Anti-CA I autoantibodies Subject RIV: EC - Immunology Impact factor: 2.367, year: 2013

Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

Science.gov (United States)

Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

2016-01-01

Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

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.

Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

Science.gov (United States)

Ship, Noam J.; Zamble, Deborah B.

2005-01-01

The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

Investigating the adduct formation of organic mercury species with carbonic anhydrase and hemoglobin from human red blood cell hemolysate by means of LC/ESI-TOF-MS and LC/ICP-MS.

Science.gov (United States)

Hogeback, Jens; Schwarzer, Miriam; Wehe, Christoph A; Sperling, Michael; Karst, Uwe

2016-01-01

The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.

Effect of carbonic anhydrase on silicate weathering and carbonate formation at present day CO2 concentrations compared to primordial values

Science.gov (United States)

Xiao, Leilei; Lian, Bin; Hao, Jianchao; Liu, Congqiang; Wang, Shijie

2015-01-01

It is widely recognized that carbonic anhydrase (CA) participates in silicate weathering and carbonate formation. Nevertheless, it is still not known if the magnitude of the effect produced by CA on surface rock evolution changes or not. In this work, CA gene expression from Bacillus mucilaginosus and the effects of recombination protein on wollastonite dissolution and carbonate formation under different conditions are explored. Real-time fluorescent quantitative PCR was used to explore the correlation between CA gene expression and sufficiency or deficiency in calcium and CO2 concentration. The results show that the expression of CA genes is negatively correlated with both CO2 concentration and ease of obtaining soluble calcium. A pure form of the protein of interest (CA) is obtained by cloning, heterologous expression, and purification. The results from tests of the recombination protein on wollastonite dissolution and carbonate formation at different levels of CO2 concentration show that the magnitudes of the effects of CA and CO2 concentration are negatively correlated. These results suggest that the effects of microbial CA in relation to silicate weathering and carbonate formation may have increased importance at the modern atmospheric CO2 concentration compared to 3 billion years ago. PMID:25583135

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

Directory of Open Access Journals (Sweden)

Dalila Jacqueline Escudero-Almanza

2012-03-01

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

Extending the scope of amantadine drug by incorporation of phenolic azo Schiff bases as potent selective inhibitors of carbonic anhydrase II, drug likeness and binding analysis.

Science.gov (United States)

Channar, Pervaiz Ali; Saeed, Aamer; Shahzad, Danish; Larik, Fayaz Ali; Hassan, Mubashir; Raza, Hussain; Abbas, Qamar; Seo, Sung-Yum

2018-05-16

A series of Amantadine based azo Schiff base dyes 6a-6e have been synthesized and characterized by 1 H NMR and 13 C NMR and evaluated for their in vitro carbonic anhydrase II inhibition activity and antioxidant activity. All of the synthesized showed excellent carbonic inhibition. Compound 6b was found to be the most potent derivative in the series, the IC 50 of 6b was found to be 0.0849 ± 0.00245μM (standard Acetazolamide IC 50 =0.9975±0.049μM). The binding interactions of the most active analogs were confirmed through molecular docking studies. Docking studies showed 6b is interacting by making two hydrogen bonds w at His93 and Ser1 residues respectively. All compounds showed a good drug score and followed Lipinski's rule. In summary, our studies have shown that these amantadine derived phenolic azo Schiff base derivatives are a new class of carbonic anhydrase II inhibitors. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Vijayakumar Somalinga

Full Text Available 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.

Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation

DEFF Research Database (Denmark)

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

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...... 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...

Characterization and function of carbonic anhydrases in the zooxanthellae-giant clam symbiosis.

Science.gov (United States)

Baillie, B K; Yellowlees, D

1998-03-22

Carbonic anhydrase (CA) has been purified from the host tissue of Tridacna gigas, a clam that lives in symbiosis with the dinoflagellate alga, Symbiodinium. At least two isoforms of CA were identified in both gill and mantle tissue. The larger (70 kDa) isoform is a glycoprotein with both N- and O-glycans attached and has highest homology to CAII. It is associated with the membrane fraction while the smaller (32 kDa) is present in the aqueous phase in both tissues. The 32 kDa CA has high homology with mammalian CAI at the N-terminus. Both isoforms cross-reacted with antibodies to CAII from chicken. Immunohistology demonstrated that the 70 kDa CA is present within the ciliated branchial filaments and cells lining the tertiary water channels in the gills of T. gigas. This is consistent with a role in the transport of inorganic carbon (Ci) to the haemolymph and therefore supply of Ci to the zooxanthellae. CA was also detected in mantle epithelial cells where it may also contribute to Ci supply to the zooxanthellae. The hyaline body and nerve tissue in the mantle express the 70 kDa CA where it may be involved in light sensing and nervous transmission.

The filamentous ascomycete Sordaria macrospora can survive in ambient air without carbonic anhydrases.

Science.gov (United States)

Lehneck, Ronny; Elleuche, Skander; Pöggeler, Stefanie

2014-06-01

The rapid interconversion of carbon dioxide and bicarbonate (hydrogen carbonate) is catalysed by metalloenzymes termed carbonic anhydrases (CAs). CAs have been identified in all three domains of life and can be divided into five evolutionarily unrelated classes (α, β, γ, δ and ζ) that do not share significant sequence similarities. The function of the mammalian, prokaryotic and plant α-CAs has been intensively studied but the function of CAs in filamentous ascomycetes is mostly unknown. The filamentous ascomycete Sordaria macrospora codes for four CAs, three of the β-class and one of the α-class. Here, we present a functional analysis of CAS4, the S. macrospora α-class CA. The CAS4 protein was post-translationally glycosylated and secreted. The knockout strain Δcas4 had a significantly reduced rate of ascospore germination. To determine the cas genes required for S. macrospora growth under ambient air conditions, we constructed double and triple mutations of the four cas genes in all possible combinations and a quadruple mutant. Vegetative growth rate of the quadruple mutant lacking all cas genes was drastically reduced compared to the wild type and invaded the agar under normal air conditions. Likewise the fruiting bodies were embedded in the agar and completely devoid of mature ascospores. © 2014 John Wiley & Sons Ltd.

Reversible pH-dependent activation/inactivation of CF(1-ATPase of spinach chloroplasts

Directory of Open Access Journals (Sweden)

A. P. Khomochkin

2017-08-01

Full Text Available The aim of the work was to study the reverse pH-dependent regulation of the enzymatic activity of the catalytic part of ATP synthase (EC 3.6.3.14 of chloroplast – coupling factor CF1. It was shown that the short-term incubation of isolated CF1 in the media with pH 4.5 or 3.5 leads to inactivation of Ca2+-ATPase, which is rapidly (t1/2 ~ 1 min restored in the medium containing 0.5-10 mM bicarbonate at pH 7.8. After acid treatment, the rate of Mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С. The increase in Ca2+– and Mg2+-АТР activity of CF1 associated with the addition of NaHCO3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. The obtained results suggest the existence of the bound bicarbonate in the CF1 structure, which apparently participates in proton transfer.

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.

Suitability of the alkalistable carbonic anhydrase from a polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic carbon sequestration.

Science.gov (United States)

Bose, Himadri; Satyanarayana, T

2016-10-01

Carbonic anhydrase (CA) was produced from the polyextremophilic (halotolerant, moderately thermophilic and alkaliphilic) bacterium Aeribacillus pallidus TSHB1 isolated from water and sediment samples of Choti Anhoni hot spring of Pipariya, Madhya Pradesh (India), is being reported to be suitable for carbon sequestration. Growth and CA production were inhibited at higher CO2 concentration (5-10 %). Under optimized culture variables (tryptone 0.8 %, yeast extract 0.08 %, glucose 1 %, micronutrient solution 1 %, inoculums size 1.10 %, agitation 200 at pH 8, and temperature 55 °C), 3.7-fold higher CA production was attained than that under unoptimized conditions. The zymogram analysis of the partially purified CA revealed an activity band corresponding to 32 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 with T 1/2 of 40, 15, and 8 min at 60, 70, and 80 °C, respectively. The CA of A. pallidus displayed a marked enhancement in the rate of CaCO3 precipitation from aqueous CO2. The CA-aided formation of CaCO3 was 42.5 mg mg(-1) protein. Scanning electron microscopy revealed the formation of rhomboid calcite crystals. This is the first report on the production and applicability of CA from the polyextremophilic A. pallidus in carbon sequestration.

Carbonic anhydrase inhibition boosts the antitumor effects of Imatinib mesylate via potentiating the antiangiogenic and antimetastatic machineries

Energy Technology Data Exchange (ETDEWEB)

Abd-El Fattah, Amal A. [Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo 11562 (Egypt); Darwish, Hebatallah A. [Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo 11562 (Egypt); Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University, Cairo (Egypt); Fathy, Nevine, E-mail: nevine.abdallah@pharma.cu.edu.eg [Department of Biochemistry, Faculty of Pharmacy, Cairo University, Cairo 11562 (Egypt); Shouman, Samia A. [Department of Cancer Biology, National Cancer Institute, Cairo University, Cairo 11796 (Egypt)

2017-02-01

Carbonic anhydrase inhibitors have emerged in the past few years as an interesting candidate for the development of novel unconventional strategies. Despite their effect in tumor regression via inhibition of tumor acidification, their potential role is not yet fully elucidated. Herein, we investigated whether acetazolamide (AZ) could modulate imatinib (IM) anticancer activity, both in breast cancer cells (T47D) and in isolated tumor specimens of Ehrlich ascites carcinoma (EAC). The impact of this combination on angiogenesis was evidenced by decreasing PDGF-A expression and enhancing that of TSP-1. In the meantime, AZ significantly suppressed IM-induced attenuation of VEGF secretion in T47D cells, most probably due to NO inhibition. The combination also dramatically decreased the metastatic activity of T47D cells by mitigating the protein levels of MMP-2 and -9 and phosphorylation of p38 MAPK, while increasing the expression of TIMP-1 and -2. In addition, a strong proapoptotic effect was observed in T47D cells after combining AZ and IM in terms of increased caspase-9 and -3 activities. Interestingly, these results were confirmed by the reduction in the isolated tumor volume, MVD, Ki-67 and VEGF expression. Eventually, the study provides a new therapeutic strategy for treating cancer. - Highlights: • A novel combination of imatinib and a carbonic anhydrase was studied. • The impact was evaluated in T47D cells and EAC-bearing mice. • The interaction suppressed PDGF-A and VEGF while enhanced TSP-1. • MMPs and p38 MAPK phosphorylation were suppressed while TIMPs were enhanced. • The interaction triggered caspase-9 and -3 activation.

Synthesis and biological evaluation of phloroglucinol derivatives possessing α-glycosidase, acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase inhibitory activity.

Science.gov (United States)

Burmaoglu, Serdar; Yilmaz, Ali O; Taslimi, Parham; Algul, Oztekin; Kilic, Deryanur; Gulcin, Ilhami

2018-02-01

A series of novel phloroglucinol derivatives were designed, synthesized, characterized spectroscopically and tested for their inhibitory activity against selected metabolic enzymes, including α-glycosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCA I and II). These compounds displayed nanomolar inhibition levels and showed K i values of 1.14-3.92 nM against AChE, 0.24-1.64 nM against BChE, 6.73-51.10 nM against α-glycosidase, 1.80-5.10 nM against hCA I, and 1.14-5.45 nM against hCA II. © 2018 Deutsche Pharmazeutische Gesellschaft.

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties.

Science.gov (United States)

De Vita, Daniela; Angeli, Andrea; Pandolfi, Fabiana; Bortolami, Martina; Costi, Roberta; Di Santo, Roberto; Suffredini, Elisabetta; Ceruso, Mariangela; Del Prete, Sonia; Capasso, Clemente; Scipione, Luigi; Supuran, Claudiu T

2017-12-01

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.

Mapping of carbonic anhydrase and estrone sulphatase in rat brain using 16-α-[18F]fluoroestradiol-3,17-β-disulphamate ([18F]FESDS)

International Nuclear Information System (INIS)

Rodig, H.; Brust, P.; Bergmann, R.; Roemer, J.; Fuechtner, F.; Steinbach, J.; Kasch, H.

2002-01-01

16α-[ 18 F]Fluoroestradiol-3,17β-disulphamate ([ 18 F]FESDS) was recently found to display affinities to carbonic anhydrase (CA) and estrone sulphatase (ES), enzymes which are expressed in the CNS and probably play a regulatory role in various brain diseases. In this study the radioligand was used to provide quantitative data on the regional distribution of these enzymes in the rat brain. (orig.)

The Evolutionary History of Daphniid α-Carbonic Anhydrase within Animalia

Science.gov (United States)

Culver, Billy W.; Morton, Philip K.

2015-01-01

Understanding the mechanisms that drive acid-base regulation in organisms is important, especially for organisms in aquatic habitats that experience rapidly fluctuating pH conditions. Previous studies have shown that carbonic anhydrases (CAs), a family of zinc metalloenzymes, are responsible for acid-base regulation in many organisms. Through the use of phylogenetic tools, this present study attempts to elucidate the evolutionary history of the α-CA superfamily, with particular interest in the emerging model aquatic organism Daphnia pulex. We provide one of the most extensive phylogenies of the evolution of α-CAs, with the inclusion of 261 amino acid sequences across taxa ranging from Cnidarians to Homo sapiens. While the phylogeny supports most of our previous understanding on the relationship of how α-CAs have evolved, we find that, contrary to expectations, amino acid conservation with bacterial α-CAs supports the supposition that extracellular α-CAs are the ancestral state of animal α-CAs. Furthermore, we show that two cytosolic and one GPI-anchored α-CA in Daphnia genus have homologs in sister taxa that are possible candidate genes to study for acid-base regulation. In addition, we provide further support for previous findings of a high rate of gene duplication within Daphnia genus, as compared with other organisms. PMID:25893130

Carbonic anhydrase I in a cartilaginous fish, the shortspine spurdog ( Squalus mitsukurii)

Science.gov (United States)

Kim, Soo Cheol; Sumi, Kanij Rukshana; Kim, Jung Woo; Choi, Myeong Rak; Min, Byung Hwa; Kho, Kang Hee

2016-09-01

Carbonic anhydrase (CA), a ubiquitous enzyme found in many species, including fishes, is involved in physiological functions such as pH homeostasis, calcification, photosynthesis, and ionic regulation. CA I, a member of the α-CA family, is a cytoplasmic isozyme involved in carbon dioxide transport, ion exchange, and acid-base balance. Approximately half of the extant shark species occur only in deep waters; however, few published studies on sharks include these taxa. As fisheries worldwide enter deeper waters, the provision of biological data for these little-known taxa is critical to their management and conservation. To address this limitation, we aimed to detect CA I in various tissues of the shortspine spurdog ( Squalus mitsukurii) and characterize its physicochemical properties by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and isoelectric focusing, together with immunohistochemistry. CA I was detected on SDS-PAGE and western blot analysis as a specific band at 29 kDa in various tissues of the shortspine spurdog, and as a specific band at pI 6.5 in various tissues of the shortspine spurdog by IEF and western blot analysis. CA I immunoreactivity in various tissues of the shortspine spurdog was detected in intracellular locations. To our knowledge, this is the first report of the localization of CA isozymes in various tissues of S. mitsukurii.

GdnHCl-induced unfolding intermediate in the mitochondrial carbonic anhydrase VA.

Science.gov (United States)

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

2016-10-01

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25°C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25M to 3.40M of the denaturant. However, CAVA gets completely denatured at 4.0M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40ns were carried out and a well-defined correlation was established for both in vitro and in silico studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Urinary carbonic anhydrase VI as a biomarker for kidney disease in pigs.

Science.gov (United States)

Nishita, Toshiho; Yatsu, Juro; Watanabe, Kazuo; Ochiai, Hideharu; Ichihara, Nobutsune; Orito, Kensuke; Arishima, Kazuyoshi

2014-11-01

This study investigated whether carbonic anhydrase (CA)-VI has utility as a biomarker in swine kidney disease. Serum chemistry, histopathology, immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) analyses were performed. In the kidney of normal healthy pigs, CA-VI was localized in the epithelial cells of the renal distal straight tubules. CA-VI levels were 16 ± 35 ng/g wet tissue and 50 ± 66 ng/mL in normal pig kidney and urine, respectively, and 136 ± 173 ng/mL in the urine of pigs with kidney disease. CA-VI urinary concentration was not correlated with urinary urea nitrogen (UUN), urinary creatinine (Cre), or urinary albumin levels in pigs with kidney disease. However, UUN and Cre levels were positively correlated in the urine of pigs with kidney disease. These data suggest that urinary CA-VI may represent a biomarker for kidney disease in pigs, particularly for disorders affecting distal straight tubules. Copyright © 2014 Elsevier Ltd. All rights reserved.

An update on anticancer drug development and delivery targeting carbonic anhydrase IX

Directory of Open Access Journals (Sweden)

Justina Kazokaitė

2017-11-01

Full Text Available The expression of carbonic anhydrase (CA IX is up-regulated in many types of solid tumors in humans under hypoxic and acidic microenvironment. Inhibition of CA IX enzymatic activity with selective inhibitors, antibodies or labeled probes has been shown to reverse the acidic environment of solid tumors and reduce the tumor growth establishing the significant role of CA IX in tumorigenesis. Thus, the development of potent antitumor drugs targeting CA IX with minimal toxic effects is important for the target-specific tumor therapy. Recently, several promising antitumor agents against CA IX have been developed to treat certain types of cancers in combination with radiation and chemotherapy. Here we review the inhibition of CA IX by small molecule compounds and monoclonal antibodies. The methods of enzymatic assays, biophysical methods, animal models including zebrafish and Xenopus oocytes, and techniques of diagnostic imaging to detect hypoxic tumors using CA IX-targeted conjugates are discussed with the aim to overview the recent progress related to novel therapeutic agents that target CA IX in hypoxic tumors.

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

Science.gov (United States)

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

2010-07-04

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 (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-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 beta-CA and two gamma-CAs. One of the putative gamma-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-gamma-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. This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-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.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

Science.gov (United States)

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

2016-07-01

Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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

Science.gov (United States)

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

2015-05-15

A new series of dithiocarbamates (DTCs) was prepared from primary/secondary amines incorporating amino/hydroxyl-alkyl, mono- and bicyclic aliphatic ring systems based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, and carbon disulfide. The compounds were investigated for the inhibition of four mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, that is, the human (h) hCA I, II, IX and XII, drug targets for antiglaucoma (hCA II and XII) or antitumor (hCA IX/XII) agents. The compounds were moderate or inefficient hCA I inhibitors (off-target isoform for both applications), efficiently inhibited hCA II, whereas some of them were low nanomolar/subnanomolar hCA IX/XII inhibitors. One DTC showed excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, with a two times better efficiency compared to the clinically used sulfonamide dorzolamide. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

Preliminary results with a torsion microbalance indicate that carbon dioxide and exposed carbonic anhydrase in the organic matrix are the basis of calcification on the skeleton surface of living corals

Directory of Open Access Journals (Sweden)

Ian M Sandeman

2012-03-01

Full Text Available Ocean acidification is altering the calcification of corals, but the mechanism is still unclear. To explore what controls calcification, small pieces from the edges of thin plates of Agaricia agaricites were suspended from a torsion microbalance into gently stirred, temperaturecontrolled, seawater. Net calcification rates were monitored while light, temperature and pH were manipulated singly. The living coral pieces were sensitive to changes in conditions, especially light, and calcification was often suspended for one or two hours or overnight. The mean calcification rate increased from 0.06 in the dark to 0.10 mg.h-1.cm-2 (T test, n=8, p<0.01 in low light (15 μmol.s-1.m-2 and showed a positive linear relationship with temperature. With a reduction of mean pH from 8.2 to 7.6 the mean calcification rate in the light (65 μmol.s-1.m-2 increased from 0.19 to 0.28 mg.h-1.cm-2 (T test, n=8, p<0.05 indicating a dependency on carbon dioxide. After waterpiking and exposure of the skeletal surface/organic matrix to seawater, calcification showed an astonishing initial increase of more than an order of magnitude then decreased following a non-linear generalised Michaelis-Menten growth curve and reached a steady rate. Calcification rate of the freshly waterpiked coral was not influenced by light and was positively correlated with temperature. For a mean pH reduction from 8.1 to 7.6 the mean calcification rate increased from 0.18 to 0.32 mg.h-1.cm-2 (T test, n=11, p<0.02 again indicating a dependency on carbon dioxide. Calcification ceased in the presence of the carbonic anhydrase inhibitor azolamide. Staining confirmed the presence of carbonic anhydrase, particularly on the ridges of septae. After immersion of waterpiked corals in seawater for 48 hours weight gain and loss became linear and positively correlated to temperature. When the mean pH was reduced from 8.2 to 7.5 the mean rate of weight gain decreased from 0.25 to 0.13 mg.h-1.cm-2 (T test, n=6

[THE EFFECT OF ACID RAIN ON ULTRASTRUCTURE AND FUNCTIONAL PARAMETERS OF PHOTOSYNTHETIC APPARATUS OF PEA LEAVES].

Science.gov (United States)

Polishchuk, A V; Vodka, M V; Belyavskaya, N A; Khomochkin, A P; Zolotareva, E K

2016-01-01

The effects of simulated acid rain (SAR) on the ultrastructure and functional parameters of the photosynthetic apparatus were studied using 14-day-old pea leaves as test system. Pea plants were sprayed with an aqueous solution containing NaNO₃(0.2 mM) and Na₂SO₄(0.2 mM) (pH 5.6, a control variant), or with the same solution, which was acidified to pH 2.5 (acid variant). Functional characteristics were determined by chlorophyll fluorescence analysis. Acid rain application caused reduction in the efficiency of the photosynthetic electron transport by 25%, which was accompanied by an increase by 85% in the quantum yield of thermal dissipation of excess light quanta. Ultrastructural changes in chloroplast were registered by transmission electron microscopy (TEM) after two days of the SAR-treatment of pea leaves. In this case, the changes in the structure of grana, heterogeneity of thylakoids packaging in granum, namely, the increase of intra-thylakoid gaps and thickness of granal thylakoids compared to the control were found. The migration of protein complexes in thylakoid membranes of chloroplasts isolated from leaves treated with SAR was suppressed. It was shown also that carbonic anhydrase activity was inhibited in chloroplast preparations isolated from SAR-treated pea leaves. We proposed a hypothesis on the possible inactivation of thylakoid carbonic anhydrase under SAR and its involvement in the inhibition of photochemical activity of chloroplasts. The data obtained allows to suggest that acid rains negatively affect the photosynthetic apparatus disrupting the membrane system of chloroplast.

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

Directory of Open Access Journals (Sweden)

Nurcan Berber

2013-01-01

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

Acidic sweep gas with carbonic anhydrase coated hollow fiber membranes synergistically accelerates CO2 removal from blood.

Science.gov (United States)

Arazawa, D T; Kimmel, J D; Finn, M C; Federspiel, W J

2015-10-01

The use of extracorporeal carbon dioxide removal (ECCO2R) is well established as a therapy for patients suffering from acute respiratory failure. Development of next generation low blood flow (carbonic anhydrase (CA) immobilized bioactive hollow fiber membrane (HFM) which significantly accelerates CO2 removal from blood in model gas exchange devices by converting bicarbonate to CO2 directly at the HFM surface. This present study tested the hypothesis that dilute sulfur dioxide (SO2) in oxygen sweep gas could further increase CO2 removal by creating an acidic microenvironment within the diffusional boundary layer adjacent to the HFM surface, facilitating dehydration of bicarbonate to CO2. CA was covalently immobilized onto poly (methyl pentene) (PMP) HFMs through glutaraldehyde activated chitosan spacers, potted in model gas exchange devices (0.0151 m(2)) and tested for CO2 removal rate with oxygen (O2) sweep gas and a 2.2% SO2 in oxygen sweep gas mixture. Using pure O2 sweep gas, CA-PMP increased CO2 removal by 31% (258 mL/min/m(2)) compared to PMP (197 mL/min/m(2)) (Premoval by 17% (230 mL/min/m(2)) compared to pure oxygen sweep gas control (Premoval increased by 109% (411 mL/min/m(2)) (Premoval, and when used in combination with bioactive CA-HFMs has a synergistic effect to more than double CO2 removal while maintaining physiologic pH. Through these technologies the next generation of intravascular and paracorporeal respiratory assist devices can remove more CO2 with smaller blood contacting surface areas. A clinical need exists for more efficient respiratory assist devices which utilize low blood flow rates (removal efficiency by shifting equilibrium from bicarbonate to gaseous CO2, through either a bioactive carbonic anhydrase enzyme coating or bulk blood acidification with lactic acid. In this study we demonstrate a novel approach to local blood acidification using an acidified sweep gas in combination with a bioactive coating to more than double CO2 removal

Carbonic anhydrases and their functional differences in human and mouse sperm physiology.

Science.gov (United States)

José, O; Torres-Rodríguez, P; Forero-Quintero, L S; Chávez, J C; De la Vega-Beltrán, J L; Carta, F; Supuran, C T; Deitmer, J W; Treviño, C L

2015-12-25

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Carbonic anhydrase 2-like in the giant clam, Tridacna squamosa: characterization, localization, response to light, and possible role in the transport of inorganic carbon from the host to its symbionts.

Science.gov (United States)

Ip, Yuen K; Koh, Clarissa Z Y; Hiong, Kum C; Choo, Celine Y L; Boo, Mel V; Wong, Wai P; Neo, Mei L; Chew, Shit F

2017-12-01

The fluted giant clam, Tridacna squamosa , lives in symbiosis with zooxanthellae which reside extracellularly inside a tubular system. Zooxanthellae fix inorganic carbon (C i ) during insolation and donate photosynthate to the host. Carbonic anhydrases catalyze the interconversion of CO 2 and HCO3-, of which carbonic anhydrase 2 (CA2) is the most ubiquitous and involved in many biological processes. This study aimed to clone a CA2 homolog ( CA2-like ) from the fleshy and colorful outer mantle as well as the thin and whitish inner mantle of T. squamosa , to determine its cellular and subcellular localization, and to examine the effects of light exposure on its gene and protein expression levels. The cDNA coding sequence of CA2-like from T. squamosa comprised 789 bp, encoding 263 amino acids with an estimated molecular mass of 29.6 kDa. A phenogramic analysis of the deduced CA2-like sequence denoted an animal origin. CA2-like was not detectable in the shell-facing epithelium of the inner mantle adjacent to the extrapallial fluid. Hence, CA2-like is unlikely to participate directly in light-enhanced calcification. By contrast, the outer mantle, which contains the highest density of tertiary tubules and zooxanthellae, displayed high level of CA2-like expression, and CA2-like was localized to the tubule epithelial cells. More importantly, exposure to light induced significant increases in the protein abundance of CA2-like in the outer mantle. Hence, CA2-like could probably take part in the increased supply of inorganic carbon (C i ) from the host clam to the symbiotic zooxanthellae when the latter conduct photosynthesis to fix C i during light exposure. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

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

Science.gov (United States)

2010-01-01

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. PMID:20598158

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.

Characterization and expression of the maize β-carbonic anhydrase gene repeat regions.

Science.gov (United States)

Tems, Ursula; Burnell, James N

2010-12-01

In maize, carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the first reaction of the C(4) photosynthetic pathway; it catalyzes the hydration of CO(2) to bicarbonate and provides an inorganic carbon source for the primary carboxylation reaction catalyzed by phosphoenolpyruvate (PEP) carboxylase. The β-CA isozymes from maize, as well as other agronomically important NADP-malic enzyme (NADP-ME) type C(4) crops, have remained relatively uncharacterized but differ significantly from the β-CAs of other C(4) monocot species primarily due to transcript length and the presence of repeat sequences. This research confirmed earlier findings of repeat sequences in maize CA transcripts, and demonstrated that the gene encoding these transcripts is also composed of repeat sequences. One of the maize CA genes was sequenced and found to encode two domains, with distinct groups of exons corresponding to the repeat regions of the transcript. We have also shown that expression of a single repeat region of the CA transcript produced active enzyme that associated as a dimer and was composed primarily of α-helices, consistent with that observed for other plant CAs. As the presence of repeat regions in the CA gene is unique to NADP-ME type C(4) monocot species, the implications of these findings in the context of the evolution of the location and function of this C(4) pathway enzyme are strongly suggestive of CA gene duplication resulting in an evolutionary advantage and a higher photosynthetic efficiency. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

Science.gov (United States)

Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

2016-04-01

Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

Nitrogen control of chloroplast development: Progress report

International Nuclear Information System (INIS)

Schmidt, G.W.

1987-11-01

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

Nitrogen control of chloroplast development: Progress report

Energy Technology Data Exchange (ETDEWEB)

Schmidt, G.W.

1987-11-01

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

Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

Science.gov (United States)

Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

2013-06-01

Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa

Science.gov (United States)

Rebello, George; Ramesar, Rajkumar; Vorster, Alvera; Roberts, Lisa; Ehrenreich, Liezle; Oppon, Ekow; Gama, Dumisani; Bardien, Soraya; Greenberg, Jacquie; Bonapace, Giuseppe; Waheed, Abdul; Shah, Gul N.; Sly, William S.

2004-01-01

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa. PMID:15090652

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.

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

Science.gov (United States)

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

2016-08-23

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 properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

DEFF Research Database (Denmark)

Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos

2016-01-01

The chloroplasts found in plants and algae, and photosynthetic microorganisms such as cyanobacteria, are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused...... on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals, as well as complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression...... of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the production levels to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons...

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....

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

Involvement of β-carbonic anhydrase (β-CA) genes in bacterial genomic islands and horizontal transfer to protists.

Science.gov (United States)

Zolfaghari Emameh, Reza; Barker, Harlan R; Hytönen, Vesa P; Parkkila, Seppo

2018-05-25

Genomic islands (GIs) are a type of mobile genetic element (MGE) that are present in bacterial chromosomes. They consist of a cluster of genes which produce proteins that contribute to a variety of functions, including, but not limited to, regulation of cell metabolism, anti-microbial resistance, pathogenicity, virulence, and resistance to heavy metals. The genes carried in MGEs can be used as a trait reservoir in times of adversity. Transfer of genes using MGEs, occurring outside of reproduction, is called horizontal gene transfer (HGT). Previous literature has shown that numerous HGT events have occurred through endosymbiosis between prokaryotes and eukaryotes.Beta carbonic anhydrase (β-CA) enzymes play a critical role in the biochemical pathways of many prokaryotes and eukaryotes. We have previously suggested horizontal transfer of β-CA genes from plasmids of some prokaryotic endosymbionts to their protozoan hosts. In this study, we set out to identify β-CA genes that might have transferred between prokaryotic and protist species through HGT in GIs. Therefore, we investigated prokaryotic chromosomes containing β-CA-encoding GIs and utilized multiple bioinformatics tools to reveal the distinct movements of β-CA genes among a wide variety of organisms. Our results identify the presence of β-CA genes in GIs of several medically and industrially relevant bacterial species, and phylogenetic analyses reveal multiple cases of likely horizontal transfer of β-CA genes from GIs of ancestral prokaryotes to protists. IMPORTANCE The evolutionary process is mediated by mobile genetic elements (MGEs), such as genomic islands (GIs). A gene or set of genes in the GIs are exchanged between and within various species through horizontal gene transfer (HGT). Based on the crucial role that GIs can play in bacterial survival and proliferation, they were introduced as the environmental- and pathogen-associated factors. Carbonic anhydrases (CAs) are involved in many critical

Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase--a new reductase.

Science.gov (United States)

Jing, Qing; Okrasa, Krzysztof; Kazlauskas, Romas J

2009-01-01

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.

Designing of phenol-based β-carbonic anhydrase1 inhibitors through QSAR, molecular docking, and MD simulation approach.

Science.gov (United States)

Ahamad, Shahzaib; Hassan, Md Imtaiyaz; Dwivedi, Neeraja

2018-05-01

Tuberculosis (Tb) is an airborne infectious disease caused by Mycobacterium tuberculosis. Beta-carbonic anhydrase 1 ( β-CA1 ) has emerged as one of the potential targets for new antitubercular drug development. In this work, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking, and molecular dynamics (MD) simulation approaches were performed on a series of natural and synthetic phenol-based β-CA1 inhibitors. The developed 3D-QSAR model ( r 2  = 0.94, q 2  = 0.86, and pred_r 2  = 0.74) indicated that the steric and electrostatic factors are important parameters to modulate the bioactivity of phenolic compounds. Based on this indication, we designed 72 new phenolic inhibitors, out of which two compounds (D25 and D50) effectively stabilized β-CA1 receptor and, thus, are potential candidates for new generation antitubercular drug discovery program.

The role of soil pH on soil carbonic anhydrase activity

Science.gov (United States)

Sauze, Joana; Jones, Sam P.; Wingate, Lisa; Wohl, Steven; Ogée, Jérôme

2018-01-01

Carbonic anhydrases (CAs) are metalloenzymes present in plants and microorganisms that catalyse the interconversion of CO2 and water to bicarbonate and protons. Because oxygen isotopes are also exchanged during this reaction, the presence of CA also modifies the contribution of soil and plant CO18O fluxes to the global budget of atmospheric CO18O. The oxygen isotope signatures (δ18O) of these fluxes differ as leaf water pools are usually more enriched than soil water pools, and this difference is used to partition the net CO2 flux over land into soil respiration and plant photosynthesis. Nonetheless, the use of atmospheric CO18O as a tracer of land surface CO2 fluxes requires a good knowledge of soil CA activity. Previous studies have shown that significant differences in soil CA activity are found in different biomes and seasons, but our understanding of the environmental and ecological drivers responsible for the spatial and temporal patterns observed in soil CA activity is still limited. One factor that has been overlooked so far is pH. Soil pH is known to strongly influence microbial community composition, richness and diversity in addition to governing the speciation of CO2 between the different carbonate forms. In this study we investigated the CO2-H2O isotopic exchange rate (kiso) in six soils with pH varying from 4.5 to 8.5. We also artificially increased the soil CA concentration to test how pH and other soil properties (texture and phosphate content) affected the relationship between kiso and CA concentration. We found that soil pH was the primary driver of kiso after CA addition and that the chemical composition (i.e. phosphate content) played only a secondary role. We also found an offset between the δ18O of the water pool with which CO2 equilibrates and total soil water (i.e. water extracted by vacuum distillation) that varied with soil texture. The reasons for this offset are still unknown.

The role of soil pH on soil carbonic anhydrase activity

Directory of Open Access Journals (Sweden)

J. Sauze

2018-01-01

Full Text Available Carbonic anhydrases (CAs are metalloenzymes present in plants and microorganisms that catalyse the interconversion of CO2 and water to bicarbonate and protons. Because oxygen isotopes are also exchanged during this reaction, the presence of CA also modifies the contribution of soil and plant CO18O fluxes to the global budget of atmospheric CO18O. The oxygen isotope signatures (δ18O of these fluxes differ as leaf water pools are usually more enriched than soil water pools, and this difference is used to partition the net CO2 flux over land into soil respiration and plant photosynthesis. Nonetheless, the use of atmospheric CO18O as a tracer of land surface CO2 fluxes requires a good knowledge of soil CA activity. Previous studies have shown that significant differences in soil CA activity are found in different biomes and seasons, but our understanding of the environmental and ecological drivers responsible for the spatial and temporal patterns observed in soil CA activity is still limited. One factor that has been overlooked so far is pH. Soil pH is known to strongly influence microbial community composition, richness and diversity in addition to governing the speciation of CO2 between the different carbonate forms. In this study we investigated the CO2–H2O isotopic exchange rate (kiso in six soils with pH varying from 4.5 to 8.5. We also artificially increased the soil CA concentration to test how pH and other soil properties (texture and phosphate content affected the relationship between kiso and CA concentration. We found that soil pH was the primary driver of kiso after CA addition and that the chemical composition (i.e. phosphate content played only a secondary role. We also found an offset between the δ18O of the water pool with which CO2 equilibrates and total soil water (i.e. water extracted by vacuum distillation that varied with soil texture. The reasons for this offset are still unknown.

Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia

Directory of Open Access Journals (Sweden)

Ahmet Menteşe

2017-12-01

Full Text Available Objective: Cancer, one of the principal causes of death, is a global social health problem. Autoantibodies developed against the organism’s self-antigens are detected in the sera of subjects with cancer. In recent years carbonic anhydrase (CA I and II autoantibodies have been shown in some autoimmune diseases and carcinomas, but the mechanisms underlying this immune response have not yet been explained. The aim of this study was to evaluate CA I and II autoantibodies in patients with acute myeloid leukemia (AML and to provide a novel perspective regarding the autoimmune basis of the disease. Materials and Methods: Anti-CA I and II antibody levels were investigated using ELISA in serum samples from 30 patients with AML and 30 healthy peers. Results: Anti-CA I and II antibody titers in the AML group were significantly higher compared with the control group (p=0.0001 and 0.018, respectively. A strong positive correlation was also determined between titers of anti-CA I and II antibodies (r=0.613, p=0.0001. Conclusion: Our results suggest that these autoantibodies may be involved in the pathogenesis of AML. More extensive studies are now needed to reveal the entire mechanism.

Crystal structures of two tetrameric β-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora.

Science.gov (United States)

Lehneck, Ronny; Neumann, Piotr; Vullo, Daniela; Elleuche, Skander; Supuran, Claudiu T; Ficner, Ralf; Pöggeler, Stefanie

2014-04-01

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like β-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat /Km of CAS1: 1.30 × 10(6) m(-1) ·s(-1) ; CAS2: 1.21 × 10(6 ) m(-1) ·s(-1) ). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn(2) (+) -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. CAS1 and CAS2 bind by x-ray crystallography (View interaction) Structural data have been deposited in the Protein Data Bank database under accession numbers 4O1J for CAS1 and 4O1K for CAS2. © 2014 FEBS.

Effect of pH on structure, function, and stability of mitochondrial carbonic anhydrase VA.

Science.gov (United States)

Idrees, Danish; Shahbaaz, Mohd; Bisetty, Krishna; Islam, Asimul; Ahmad, Faizan; Hassan, Md Imtaiyaz

2017-02-01

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0-pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of k cat and k cat /K m at pH 9.0 are 3.7 × 10 6  s -1 and 5.5 × 10 7  M -1  s -1 , respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.

Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize.

Directory of Open Access Journals (Sweden)

Prakitchai Chotewutmontri

2016-07-01

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

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

Directory of Open Access Journals (Sweden)

Marcelo Zerati

2013-07-01

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

Protein import into chloroplasts requires a chloroplast ATPase

International Nuclear Information System (INIS)

Pain, D.; Blobel, G.

1987-01-01

The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

Protein import into chloroplasts requires a chloroplast ATPase

Energy Technology Data Exchange (ETDEWEB)

Pain, D.; Blobel, G.

1987-05-01

The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

Diuretics: from classical carbonic anhydrase inhibitors to novel applications of the sulfonamides.

Science.gov (United States)

Supuran, Claudiu T

2008-01-01

The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO(2)NH(2) moieties acting as an effective zinc-binding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

A systematic quantification of carbonic anhydrase transcripts in the mouse digestive system

Directory of Open Access Journals (Sweden)

Parkkila Seppo

2007-03-01

Full Text Available Abstract Background Carbonic anhydrases (CAs are physiologically important enzymes which participate in many gastrointestinal processes such as acid and bicarbonate secretion and metabolic pathways including gluconeogenesis and ureagenesis. The genomic data suggests that there are thirteen enzymatically active members of the mammalian CA isozyme family. In the present study, we systematically examined the mRNA expression levels of all known CA isozymes by quantitative real-time PCR in eight tissues of the digestive system of male and female mice. Results The CAs expressed in all tissues were Car5b, Car7, and Car15, among which Car5b showed moderate and Car7 and Car15 extremely low expression levels. Car3, Car12, Car13, and Car14 were detected in seven out of eight tissues and Car2 and Car4 were expressed in six tissues. Importantly, Car1, Car3, and Car13 showed very high expression levels in certain tissues as compared to the other CAs, suggesting that these low activity isozymes may also participate in physiological processes other than CA catalysis and high expression levels are required to fulfil their functions in the body. Conclusion A comprehensive mRNA expression profile of the 13 enzymatically active CAs in the murine gastrointestinal tract was produced in the present study. It contributes to a deeper understanding of the distribution of CA isozymes and their potential roles in the mouse digestive system.

Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

Science.gov (United States)

Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

2015-08-01

Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research

Comparative study of carbonic anhydrase activity in waters among different geological eco-environments of Yangtze River basin and its ecological significance.

Science.gov (United States)

Nzung'a, Sila Onesmus; Pan, Weizhi; Shen, Taiming; Li, Wei; Qin, Xiaoqun; Wang, Chenwei; Zhang, Liankai; Yu, Longjiang

2018-04-01

This study provides the presence of carbonic anhydrase (CA) activity in waters of the Yangtze River basin, China, as well as the correlation of CA activity with HCO 3 - concentration and CO 2 sink flux. Different degrees of CA activity could be detected in almost all of the water samples from different geological eco-environments in all four seasons. The CA activity of water samples from karst areas was significantly higher than from non-karst areas (PP3 - concentration (r=0.672, P2 sink flux (r=0.602, P=0.076) in karst areas. This suggests that CA in waters might have a promoting effect on carbon sinks for atmospheric CO 2 in karst river basins. In conditions of similar geological type, higher CA activity was generally detected in water samples taken from areas that exhibited better eco-environments, implying that the CA activity index of waters could be used as an indicator for monitoring ecological environments and protection of river basins. These findings suggest that the role of CA in waters in the karst carbon sink potential of river basins is worthy of further in-depth studies. Copyright © 2017. Published by Elsevier B.V.

Tobacco Nectarin III is a bifunctional enzyme with monodehydroascorbate reductase and carbonic anhydrase activities.

Science.gov (United States)

Carter, Clay J; Thornburg, Robert W

2004-02-01

Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequences also revealed that NEC2 is a breakdown product of NEC3. Using these N-terminal peptide sequences, degenerate oligonucleotides were designed that permitted the isolation of a partial NEC3 cDNA. This cDNA was then used to probe a nectary specific cDNA library and a full-length NEC3 cDNA clone was isolated. Complete sequence analysis confirmed the identity of NEC3 as a dioscorin-like protein. MALDI-TOF mass spectrometric fingerprinting of tryptic peptides derived from the purified NEC3 confirmed that this protein was encoded by the isolated cDNA. NEC3 was shown to possess both carbonic anhydrase and monodehydroascorbate reductase activities. RT-PCR based expression analyses demonstrated that NEC3 transcript is expressed throughout nectary development as well as in other floral organs. A proposed function in the maintenance of pH and oxidative balance in nectar is discussed.

Mechanisms of carbon dioxide acquisition and CO2 sensing in marine diatoms: a gateway to carbon metabolism.

Science.gov (United States)

Matsuda, Yusuke; Hopkinson, Brian M; Nakajima, Kensuke; Dupont, Christopher L; Tsuji, Yoshinori

2017-09-05

Diatoms are one of the most successful marine eukaryotic algal groups, responsible for up to 20% of the annual global CO 2 fixation. The evolution of a CO 2 -concentrating mechanism (CCM) allowed diatoms to overcome a number of serious constraints on photosynthesis in the marine environment, particularly low [CO 2 ] aq in seawater relative to concentrations required by the CO 2 fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which is partly due to the slow diffusion rate of CO 2 in water and a limited CO 2 formation rate from [Formula: see text] in seawater. Diatoms use two alternative strategies to take up dissolved inorganic carbon (DIC) from the environment: one primarily relies on the direct uptake of [Formula: see text] through plasma-membrane type solute carrier (SLC) 4 family [Formula: see text] transporters and the other is more reliant on passive diffusion of CO 2 formed by an external carbonic anhydrase (CA). Bicarbonate taken up into the cytoplasm is most likely then actively transported into the chloroplast stroma by SLC4-type transporters on the chloroplast membrane system. Bicarbonate in the stroma is converted into CO 2 only in close proximity to RubisCO preventing unnecessary CO 2 leakage. CAs play significant roles in mobilizing DIC as it is progressively moved towards the site of fixation. However, the evolutionary types and subcellular locations of CAs are not conserved between different diatoms, strongly suggesting that this DIC mobilization strategy likely evolved multiple times with different origins. By contrast, the recent discovery of the thylakoid luminal θ-CA indicates that the strategy to supply CO 2 to RubisCO in the pyrenoid may be very similar to that of green algae, and strongly suggests convergent coevolution in CCM function of the thylakoid lumen not only among diatoms but among eukaryotic algae in general. In this review, both experimental and corresponding theoretical models of the diatom CCMs are

Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II.

Science.gov (United States)

Gökçe, Başak; Gençer, Nahit; Arslan, Oktay; Turkoğlu, Sumeyye Aydogan; Alper, Meltem; Köçkar, Feray

2012-02-01

The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC(50) values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC(50) concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 μM and 4250 μM and for hCA II 950 μM and 6200 μM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes.

Motif analysis unveils the possible co-regulation of chloroplast genes and nuclear genes encoding chloroplast proteins.

Science.gov (United States)

Wang, Ying; Ding, Jun; Daniell, Henry; Hu, Haiyan; Li, Xiaoman

2012-09-01

Chloroplasts play critical roles in land plant cells. Despite their importance and the availability of at least 200 sequenced chloroplast genomes, the number of known DNA regulatory sequences in chloroplast genomes are limited. In this paper, we designed computational methods to systematically study putative DNA regulatory sequences in intergenic regions near chloroplast genes in seven plant species and in promoter sequences of nuclear genes in Arabidopsis and rice. We found that -35/-10 elements alone cannot explain the transcriptional regulation of chloroplast genes. We also concluded that there are unlikely motifs shared by intergenic sequences of most of chloroplast genes, indicating that these genes are regulated differently. Finally and surprisingly, we found five conserved motifs, each of which occurs in no more than six chloroplast intergenic sequences, are significantly shared by promoters of nuclear-genes encoding chloroplast proteins. By integrating information from gene function annotation, protein subcellular localization analyses, protein-protein interaction data, and gene expression data, we further showed support of the functionality of these conserved motifs. Our study implies the existence of unknown nuclear-encoded transcription factors that regulate both chloroplast genes and nuclear genes encoding chloroplast protein, which sheds light on the understanding of the transcriptional regulation of chloroplast genes.

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

Science.gov (United States)

Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

2006-12-01

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 efficient promoters 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.

Ion and metabolite transport in the chloroplast of algae: lessons from land plants.

Science.gov (United States)

Marchand, Justine; Heydarizadeh, Parisa; Schoefs, Benoît; Spetea, Cornelia

2018-06-01

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deficiency of Carbonic Anhydrase II Results in a Urinary Concentrating Defect

Directory of Open Access Journals (Sweden)

Devishree Krishnan

2018-01-01

Full Text Available Carbonic anhydrase II (CAII is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1 interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL; however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

Carbon dioxide is a powerful inducer of monokaryotic hyphae and spore development in Cryptococcus gattii and carbonic anhydrase activity is dispensable in this dimorphic transition.

Science.gov (United States)

Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

2014-01-01

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).

SYNTHESIS AND EVALUATION OF NEW PHTHALAZINE SUBSTITUTED β-LACTAM DERIVATIVES AS CARBONIC ANHYDRASE INHIBITORS.

Science.gov (United States)

Berber, Nurcan; Arslan, Mustafa; Bilen, Çiğdem; Sackes, Zübeyde; Gençer, Nahit; Arslan, Oktay

2015-01-01

A new series of phthalazine substituted β-lactam derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated. 2H-Indazolo[2,1-b]phthala- zine-trione derivative was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and the nitro group was reduced to 13-(4-aminophenyl)-3,3-dimethyl-3,4-dihydro- 2H-indazolo[1,2-b]phthalazine-1,6,11(13H)-trione with SnCl2 · 2H2O. The reduced compound was re- acted with different aromatic aldehydes, and phthalazine substituted imines were synthesized. The imine compounds undergo (2+2) cycloaddition reactions with ketenes to produce 2H-indazolo[2,1-b]phthala-zine-trione substituted β-lactam derivatives. The β-lactam compounds were tested as inhibitors of the CA isoenzyme activity. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. 1-(4-(3,3-dimethyl- 1,6,1 1-trioxo-2,3,4,6,11,13-hexahydro-1H-indazolo[1,2-b]phthalazin-13- yl)phenyl)-2-oxo-4-p-tolylazetidin-3-yl acetate (IC50 = 6.97 µM for hCA I and 8.48 µM for hCA II) had the most inhibitory effect.

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

Science.gov (United States)

Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

2015-07-01

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

Science.gov (United States)

Gilmour, K M; Perry, S F; Bernier, N J; Henry, R P; Wood, C M

2001-01-01

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

Science.gov (United States)

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Feng, Kuan; Zhang, Shuangyan; Huang, Jiefang; Miao, Xiang; Baggi, Fulvio; Ostrom, Rennolds S; Zhang, Yanyun; Chen, Xiangjun; Xu, Congfeng

2017-01-01

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

Science.gov (United States)

Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

2014-04-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice.

Science.gov (United States)

Sade, Nir; Umnajkitikorn, Kamolchanok; Rubio Wilhelmi, Maria Del Mar; Wright, Matthew; Wang, Songhu; Blumwald, Eduardo

2018-02-12

Abiotic stress-induced senescence in crops is a process particularly affecting the photosynthetic apparatus, decreasing photosynthetic activity and inducing chloroplast degradation. A pathway for stress-induced chloroplast degradation that involves the CHLOROPLAST VESICULATION (CV) gene was characterized in rice (Oryza sativa) plants. OsCV expression was up-regulated with the age of the plants and when plants were exposed to water-deficit conditions. The down-regulation of OsCV expression contributed to the maintenance of the chloroplast integrity under stress. OsCV-silenced plants displayed enhanced source fitness (i.e. carbon and nitrogen assimilation) and photorespiration, leading to water-deficit stress tolerance. Co-immunoprecipitation, intracellular co-localization, and bimolecular fluorescence demonstrated the in vivo interaction between OsCV and chloroplastic glutamine synthetase (OsGS2), affecting source-sink relationships of the plants under stress. Our results would indicate that the OsCV-mediated chloroplast degradation pathway is involved in the regulation of nitrogen assimilation during stress-induced plant senescence. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

SKL1 Is Essential for Chloroplast Development in Arabidopsis

Directory of Open Access Journals (Sweden)

Huimin Xu

2018-02-01

Full Text Available The Arabidopsis shikimate kinase-like 1 (skl1-8 mutant is characterized by a pigment-defective phenotype. Although the related phenotypical defect mainly has been attributed to the blocking of chloroplast development, the molecular functions of SKL1 remain largely unknown. In this study, we combined multiple approaches to investigate the potential functions of SKL1. Results showed that the skl1-8 mutant exhibited an albino phenotype and had dramatically reduced chlorophyll content as a consequence of a single nuclear recessive gene mutation. Chemical complementation analysis indicated that SKL1 does not function as SK enzyme in the shikimate pathway. In addition, by chlorophyll fluorescence parameters and immunoblot analysis, the levels of photosynthetic proteins are substantially reduced. Moreover, by transcriptome analysis, specific groups of nuclear genes involved in photosynthesis, such as light-harvesting complex, pigment metabolism, carbon metabolism, and chloroplast gene expression, were down-regulated, whereas several defense and oxidative stress responsive genes were up-regulated in the skl1-8 mutant compared with the wide type. Furthermore, we found the expression of genes related to auxin transport and response was repressed in the skl1-8 mutant, probable suggesting that SKL1 is involved in auxin-related pathways during chloroplast development. Together, these results provide a useful reference for characterization of SKL1 function during chloroplast biogenesis and development.

Identification and characterization of a novel zebrafish (Danio rerio pentraxin–carbonic anhydrase

Directory of Open Access Journals (Sweden)

Maarit S. Patrikainen

2017-12-01

Full Text Available Background Carbonic anhydrases (CAs are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf. The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org. The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Science.gov (United States)

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Novel 6- and 7-Substituted Coumarins with Inhibitory Action against Lipoxygenase and Tumor-Associated Carbonic Anhydrase IX

Directory of Open Access Journals (Sweden)

Aikaterini Peperidou

2018-01-01

Full Text Available A series of carboxamide derivatives of 6- and 7-substituted coumarins have been prepared by an original procedure starting from the corresponding 6- or 7-hydroxycoumarins which were alkylated with ethyl iodoacetate, and the obtained ester was converted to the corresponding carboxylic acids which were thereafter reacted with a series of aromatic/aliphatic/heterocyclic amines leading to the desired amides. The new derivatives were investigated as inhibitors of two enzymes, human carbonic anhydrases (hCAs and soy bean lipoxygenase (LOX. Compounds 4a and 4b were potent LOX inhibitors, whereas many effective hCA IX inhibitors (KIs in the range of 30.2–30.5 nM were detected in this study. Two compounds, 4b and 5b, showed the phenomenon of dual inhibition. Furthermore, these coumarins did not significantly inhibit the widespread cytosolic isoforms hCA I and II, whereas they were weak hCA IV inhibitors, making them hCA IX-selective inhibitors. As hCA IX and LOX are validated antitumor targets, these results are promising for the investigation of novel drug targets involved in tumorigenesis.

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.

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

Directory of Open Access Journals (Sweden)

Hilal Kuday

2014-01-01

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

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome.

Science.gov (United States)

Kamal, Abu Hena Mostafa; Cho, Kun; Komatsu, Setsuko; Uozumi, Nobuyuki; Choi, Jong-Soon; Woo, Sun Hee

2012-05-01

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

The ability of anti-carbonic anhydrase II antibody to distinguish autoimmune cholangitis from primary biliary cirrhosis in Japanese patients.

Science.gov (United States)

Akisawa, N; Nishimori, I; Miyaji, E; Iwasaki, S; Maeda, T; Shimizu, H; Sato, N; Onishi, S

1999-06-01

Serum antibody against carbonic anhydrase (CA) II has been described as a serological marker for distinguishing autoimmune cholangitis (AIC) from primary biliary cirrhosis (PBC). To validate this finding in a Japanese population, we evaluated sera from patients with PBC and AIC for antibody to human CA II. An enzyme-linked immunosorbent assay was employed to quantify serum antibody against CA II in patients with PBC (n = 40), AIC (n = 23), autoimmune hepatitis (n = 10), and extrahepatic obstructive jaundice (n = 10). Compared with the finding of a 4% prevalence of anti-CAII antibody in healthy subjects (n = 24), a significantly higher prevalence of anti-CA II antibody was detected in patients with PBC (35%) and AIC (30%) (P jaundice. No significant difference was observed between PBC and AIC patients. These results showed that AIC and PBC would be indistinguishable by anti-CA II antibody testing in Japanese patients. However, the finding of serum anti-CA II antibody in patients with PBC and AIC supports the disease concept of autoimmune exocrinopathy.

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment.

Science.gov (United States)

Larkin, Robert M; Stefano, Giovanni; Ruckle, Michael E; Stavoe, Andrea K; Sinkler, Christopher A; Brandizzi, Federica; Malmstrom, Carolyn M; Osteryoung, Katherine W

2016-02-23

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.

Carbonic anhydrase IX inhibition affects viability of cancer cells adapted to extracellular acidosis.

Science.gov (United States)

Andreucci, Elena; Peppicelli, Silvia; Carta, Fabrizio; Brisotto, Giulia; Biscontin, Eva; Ruzzolini, Jessica; Bianchini, Francesca; Biagioni, Alessio; Supuran, Claudiu T; Calorini, Lido

2017-12-01

Among the players of the adaptive response of cancer cells able to promote a resistant and aggressive phenotype, carbonic anhydrase IX (CAIX) recently has emerged as one of the most relevant drug targets. Indeed, CAIX targeting has received a lot of interest, and selective inhibitors are currently under clinical trials. Hypoxia has been identified as the master inductor of CAIX, but, to date, very few is known about the influence that another important characteristic of tumor microenvironment, i.e., extracellular acidosis, exerts on CAIX expression and activity. In the last decades, acidic microenvironment has been associated with aggressive tumor phenotype endowed with epithelial-to-mesenchymal transition (EMT) profile, high invasive and migratory ability, apoptosis, and drug resistance. We demonstrated that melanoma, breast, and colorectal cancer cells transiently and chronically exposed to acidified medium (pH 6.7 ± 0.1) showed a significantly increased CAIX expression compared to those grown in standard conditions (pH 7.4 ± 0.1). Moreover, we observed that the CAIX inhibitor FC16-670A (also named SLC-0111, which just successfully ended phase I clinical trials) not only prevents such increased expression under acidosis but also promotes apoptotic and necrotic programs only in acidified cancer cells. Thus, CAIX could represent a selective target of acidic cancer cells and FC16-670A inhibitor as a useful tool to affect this aggressive subpopulation characterized by conventional therapy escape. Cancer cells overexpress CAIX under transient and chronic extracellular acidosis. Acidosis-induced CAIX overexpression is NF-κB mediated and HIF-1α independent. FC16-670A prevents CAIX overexpression and induces acidified cancer cell death.

Chemical Rescue of Enzymes: Proton Transfer in Mutants of Human Carbonic Anhydrase II

Science.gov (United States)

Maupin, C. Mark; Castillo, Norberto; Taraphder, Srabani; Tu, Chingkuang; McKenna, Robert; Silverman, David N.; Voth, Gregory A.

2011-01-01

In human carbonic anhydrase II (HCA II) the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II give a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site specific mutations intended to disrupt 4MI binding have demonstrated these sites to be non-productive. In the present work MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway PMID:21452838

Diuretics with carbonic anhydrase inhibitory action: a patent and literature review (2005 - 2013).

Science.gov (United States)

Carta, Fabrizio; Supuran, Claudiu T

2013-01-01

The benzothiadiazines and high ceiling diuretics (hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide) contain primary sulfamoyl moieties acting as zinc-binding groups in the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). These drugs are widely used clinically and were recently shown to weakly inhibit isoforms CA I and II, but to possess stronger activity against isoforms involved in other important pathologies, for example, obesity, cancer, epilepsy and hypertension. The class of clinically used diuretics, with CA inhibitory properties, is the main topic of the review. A patent literature review covering the period from 2005 to 2013 is presented. This section presents an overview of the patent literature in the sulfonamide diuretic field. Most of the patents deal with the combination of diuretic sulfonamide CA inhibitors with other agents useful in the management of cardiovascular diseases and obesity. Such combinations exert a better therapeutic activity compared to similar diuretics that do not inhibit CAs, raising the question of the polypharmacological and drug repositioning effects of these old drugs. These effects seem to be due to the potent inhibition of such drugs against CA isoforms present in kidneys and blood vessels, which explain both the blood pressure lowering effects as well as organ-protective activity of the drugs. An explanation of these data is provided by the fact that inhibition of the renal CAs leads to a large increase of the nitrite excretion in urine, suggesting that renal CAs are involved in nitrite reabsorption in humans. Important lessons for the drug design of sulfonamide CA inhibitors (CAIs) can be drawn from these data.

A physically interpretable quantum-theoretic QSAR for some carbonic anhydrase inhibitors with diverse aromatic rings, obtained by a new QSAR procedure.

Science.gov (United States)

Clare, Brian W; Supuran, Claudiu T

2005-03-15

A QSAR based almost entirely on quantum theoretically calculated descriptors has been developed for a large and heterogeneous group of aromatic and heteroaromatic carbonic anhydrase inhibitors, using orbital energies, nodal angles, atomic charges, and some other intuitively appealing descriptors. Most calculations have been done at the B3LYP/6-31G* level of theory. For the first time we have treated five-membered rings by the same means that we have used for benzene rings in the past. Our flip regression technique has been expanded to encompass automatic variable selection. The statistical quality of the results, while not equal to those we have had with benzene derivatives, is very good considering the noncongeneric nature of the compounds. The most significant correlation was with charge on the atoms of the sulfonamide group, followed by the nodal orientation and the solvation energy calculated by COSMO and the charge polarization of the molecule calculated as the mean absolute Mulliken charge over all atoms.

Chloroplast Redox Poise

DEFF Research Database (Denmark)

Steccanella, Verdiana

the redox status of the plastoquinone pool and chlorophyll biosynthesis. Furthermore, in the plant cell, the equilibrium between redox reactions and ROS signals is also maintained by various balancing mechanisms among which the thioredoxin reductase-thioredoxin system (TR-Trx) stands out as a mediator......The redox state of the chloroplast is maintained by a delicate balance between energy production and consumption and is affected by the need to avoid increased production of reactive oxygen species (ROS). Redox power and ROS generated in the chloroplast are essential for maintaining physiological...... metabolic pathways and for optimizing chloroplast functions. The redox poise of photosynthetic electron transport components like plastoquinone is crucial to initiate signaling cascades and might also be involved in key biosynthetic pathways such as chlorophyll biosynthesis. We, therefore, explored...

Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

Science.gov (United States)

Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

2011-12-15

In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

Inorganic carbon acquisition in potentially toxic and non-toxic diatoms: the effect of pH-induced changes in the seawater carbonate chemistry

DEFF Research Database (Denmark)

Trimborn, S; Lundholm, Nina; Thoms, S

2008-01-01

. In terms of carbon source, all species took up both CO2 and HCO3-. K-1/2 values for inorganic carbon uptake decreased with increasing pH in two species, while in N. navis-varingica apparent affinities did not change. While the contribution of HCO3- to net fixation was more than 85% in S. stellaris......The effects of pH-induced changes in seawater carbonate chemistry on inorganic carbon (C-i) acquisition and domoic acid (DA) production were studied in two potentially toxic diatom species, Pseudo-nitzschia multiseries and Nitzschia navis-varingica, and the non-toxic Stellarima stellaris. In vivo...... activities of carbonic anhydrase (CA), photosynthetic O-2 evolution and CO2 and HCO3- uptake rates were measured by membrane inlet MS in cells acclimated to low (7.9) and high pH (8.4 or 8.9). Species-specific differences in the mode of carbon acquisition were found. While extracellular carbonic anhydrase (e...

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

International Nuclear Information System (INIS)

Yoo, Chong Woo; Nam, Byung-Ho; Kim, Joo-Young; Shin, Hye-Jin; Lim, Hyunsun; Lee, Sun; Lee, Su-Kyoung; Lim, Myong-Cheol; Song, Yong-Jung

2010-01-01

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

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

Purification and inhibition studies with anions and sulfonamides of an α-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii.

Science.gov (United States)

Cincinelli, Alessandra; Martellini, Tania; Innocenti, Alessio; Scozzafava, Andrea; Supuran, Claudiu T

2011-03-15

A high activity α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic activity for the physiologic CO(2) hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a k(cat) of 1.1×10(6) s(-1), and a k(cat)/K(m) of 1.4×10(8) M(-1) s(-1). The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (K(I)s in the range of 46-100 μM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with K(I)s in the range of 5.7-67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with K(I)s in the range of 513-5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO(2) partial pressure and hypoxic conditions in which Weddell seals spend much of their life. Copyright © 2011 Elsevier Ltd. All rights reserved.

Kinetics of CO2 diffusion in human carbonic anhydrase: a study using molecular dynamics simulations and the Markov-state model.

Science.gov (United States)

Chen, Gong; Kong, Xian; Lu, Diannan; Wu, Jianzhong; Liu, Zheng

2017-05-10

Molecular dynamics (MD) simulations, in combination with the Markov-state model (MSM), were applied to probe CO 2 diffusion from an aqueous solution into the active site of human carbonic anhydrase II (hCA-II), an enzyme useful for enhanced CO 2 capture and utilization. The diffusion process in the hydrophobic pocket of hCA-II was illustrated in terms of a two-dimensional free-energy landscape. We found that CO 2 diffusion in hCA-II is a rate-limiting step in the CO 2 diffusion-binding-reaction process. The equilibrium distribution of CO 2 shows its preferential accumulation within a hydrophobic domain in the protein core region. An analysis of the committors and reactive fluxes indicates that the main pathway for CO 2 diffusion into the active site of hCA-II is through a binding pocket where residue Gln 136 contributes to the maximal flux. The simulation results offer a new perspective on the CO 2 hydration kinetics and useful insights toward the development of novel biochemical processes for more efficient CO 2 sequestration and utilization.

Studies on bicarbonate transporters and carbonic anhydrase in porcine non-pigmented ciliary epithelium

Science.gov (United States)

Shahidullah, Mohammad; C-H, To; Pelis, Ryan M.; Delamere, Nicholas A

2009-01-01

Purpose Bicarbonate transport plays a role in aqueous humor (AH) secretion. Here, we examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine non-pigmented ciliary epithelium (NPE). Methods Cytoplasmic pH (pHi) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC) and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. Results Anion exchanger AE2, CAII and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO2/HCO3− free HEPES buffer, exposure to a CO2/HCO3−-containing buffer caused a rapid acidification followed by a gradual pHi increase. Subsequent removal of CO2/HCO3− with HEPES buffer caused rapid alkalinization followed by gradual pHi decrease. The rate of gradual alkalinization after addition of HCO3−/CO2 was inhibited by sodium-free conditions, DIDS, CA inhibitors acetazolamide and methazolamide but not by Na-H exchange inhibitor dimethylamiloride or low chloride buffer. The phase of gradual acidification after removal of HCO3−/CO2 was inhibited by DIDS, acetazolamide, methazolamide and by low chloride buffer. DIDS reduced baseline pHi. In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44% respectively. Conclusion The results suggest the NPE uses a Na+-HCO3− cotransporter to import bicarbonate and a Cl−/HCO3− exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII and CAIV are enriched in the NPE layer of the ciliary body and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye. PMID:19011010

The role of chloroplasts in plant pathology.

Science.gov (United States)

Sowden, Robert G; Watson, Samuel J; Jarvis, Paul

2018-04-13

Plants have evolved complex tolerance systems to survive abiotic and biotic stresses. Central to these programmes is a sophisticated conversation of signals between the chloroplast and the nucleus. In this review, we examine the antagonism between abiotic stress tolerance (AST) and immunity: we propose that to generate immunogenic signals, plants must disable AST systems, in particular those that manage reactive oxygen species (ROS), while the pathogen seeks to reactivate or enhance those systems to achieve virulence. By boosting host systems of AST, pathogens trick the plant into suppressing chloroplast immunogenic signals and steer the host into making an inappropriate immune response. Pathogens disrupt chloroplast function, both transcriptionally-by secreting effectors that alter host gene expression by interacting with defence-related kinase cascades, with transcription factors, or with promoters themselves-and post-transcriptionally, by delivering effectors that enter the chloroplast or alter the localization of host proteins to change chloroplast activities. These mechanisms reconfigure the chloroplast proteome and chloroplast-originating immunogenic signals in order to promote infection. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Photosynthesis in a different light: Spectro-microscopy for in vivo characterisation of chloroplasts

Directory of Open Access Journals (Sweden)

Sébastien ePeter

2014-06-01

Full Text Available During photosynthesis, energy conversion at the two photosystems is controlled by highly complex and dynamic adaptation processes triggered by external factors such as light quality, intensity, and duration, or internal cues such as carbon availability. These dynamics have remained largely concealed so far, because current analytical techniques are based on the investigation of isolated chloroplasts lacking full adaptation ability and are performed at non-physiologically low temperatures. Here, we use non-invasive in planta spectro-microscopic approaches to investigate living chloroplasts in their native environment at ambient temperatures. This is a valuable approach to study the complex function of these systems, because an intrinsic property – the fluorescence emission – is exploited and no additional external perturbations are introduced. Our analysis demonstrates a dynamic adjustment of not only the photosystemI/photosystemII (PSI/PSII intensity ratio in the chloroplasts but also of the capacity of the LHCs for energy transfer in response to environmental and internal cues.

Direct chloroplast sequencing: comparison of sequencing platforms and analysis tools for whole chloroplast barcoding.

Directory of Open Access Journals (Sweden)

Marta Brozynska

Full Text Available Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina and Ion Torrent (Life Technology sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare. Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.

Topical carbonic anhydrase inhibitors and visual function in glaucoma and ocular hypertension.

Science.gov (United States)

Gugleta, Konstantin

2010-06-01

Dorzolamide and brinzolamide are topical carbonic anhydrase inhibitors (CAI) indicated for patients with glaucoma and ocular hypertension. An evidence-based review of clinical trials of dorzolamide and brinzolamide was undertaken to determine an effect of these medications on visual function (primarily visual field) in open-angle glaucoma and ocular hypertension. Using the keywords 'dorzolamide' and 'brinzolamide', all articles describing trials of these medications reporting on visual acuity, contrast sensitivity and visual field from September 1966 to July 2009 were found in MEDLINE and EMBASE databases. No information from other sources was included in this review. A relatively modest number of trials was identified, where impact of therapy on one or more of the visual function modes was reported. In the studies of less than 1 year duration (3 days to 1 year, 23 studies) in all but three studies treatment with topical CAIs did not influence visual function, in two studies with dorzolamide some improvement in the contrast sensitivity was observed and in one open-label retrospective no-control-group study with dorzolamide visual field indices improved significantly. A different picture was seen in long-term studies, which were designed and powered to detect changes in visual field. One large study (European Glaucoma Prevention Study) with dorzolamide versus placebo failed to detect significant protective effect of the drug on glaucoma occurrence in ocular hypertensives. Several interesting aspects of this study are discussed in detail. The other two long-term studies reported on the superiority of adding dorzolamide over timolol therapy alone, and the superiority of the combination of dorzolamide and timolol over brinzolamide and timolol in terms of improving ocular blood flow (retrobulbar Color Doppler Imaging--CDI parameters) as well as in terms of visual field preservation in glaucoma patients over 4 to 5 years. For the first time one study could demonstrate

The Cytoplasmic Carbonic Anhydrases βCA2 and βCA4 Are Required for Optimal Plant Growth at Low CO2.

Science.gov (United States)

DiMario, Robert J; Quebedeaux, Jennifer C; Longstreth, David J; Dassanayake, Maheshi; Hartman, Monica M; Moroney, James V

2016-05-01

Carbonic anhydrases (CAs) are zinc metalloenzymes that interconvert CO2 and HCO3 (-) In plants, both α- and β-type CAs are present. We hypothesize that cytoplasmic βCAs are required to modulate inorganic carbon forms needed in leaf cells for carbon-requiring reactions such as photosynthesis and amino acid biosynthesis. In this report, we present evidence that βCA2 and βCA4 are the two most abundant cytoplasmic CAs in Arabidopsis (Arabidopsis thaliana) leaves. Previously, βCA4 was reported to be localized to the plasma membrane, but here, we show that two forms of βCA4 are expressed in a tissue-specific manner and that the two proteins encoded by βCA4 localize to two different regions of the cell. Comparing transfer DNA knockout lines with wild-type plants, there was no reduction in the growth rates of the single mutants, βca2 and βca4 However, the growth rate of the double mutant, βca2βca4, was reduced significantly when grown at 200 μL L(-1) CO2 The reduction in growth of the double mutant was not linked to a reduction in photosynthetic rate. The amino acid content of leaves from the double mutant showed marked reduction in aspartate when compared with the wild type and the single mutants. This suggests the cytoplasmic CAs play an important but not previously appreciated role in amino acid biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

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

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

OpenAIRE

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacc...

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias).

Science.gov (United States)

Gilmour, K M; Bayaa, M; Kenney, L; McNeill, B; Perry, S F

2007-01-01

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.

Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein.

Science.gov (United States)

Marie, Benjamin; Luquet, Gilles; Bédouet, Laurent; Milet, Christian; Guichard, Nathalie; Medakovic, Davorin; Marin, Frédéric

2008-10-13

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.

Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

International Nuclear Information System (INIS)

Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

1986-01-01

In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

Dioscorin, the major tuber storage protein of yam (Dioscorea batatas decne) with carbonic anhydrase and trypsin inhibitor activities.

Science.gov (United States)

Hou, W C; Liu, J S; Chen, H J; Chen, T E; Chang, C F; Lin, Y H

1999-05-01

Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

Reaction Coordinate, Free Energy, and Rate of Intramolecular Proton Transfer in Human Carbonic Anhydrase II.

Science.gov (United States)

Paul, Sanjib; Paul, Tanmoy Kumar; Taraphder, Srabani

2018-03-22

The role of structure and dynamics of an enzyme has been investigated at three different stages of its function including the chemical event it catalyzes. A one-pot computational method has been designed for each of these stages on the basis of classical and/or quantum mechanical-molecular mechanical molecular dynamics and transition path sampling simulations. For a pair of initial and final states A and B separated by a high free-energy barrier, using a two-stage selection process, several collective variables (CVs) are identified that can delineate A and B. However, these CVs are found to exhibit strong cross-coupling over the transition paths. A set of mutually orthogonal order parameters is then derived from these CVs and an optimal reaction coordinate, r, determined applying half-trajectory likelihood maximization along with a Bayesian information criterion. The transition paths are also used to project the multidimensional free energy surface and barrier crossing dynamics along r. The proposed scheme has been applied to the rate-determining intramolecular proton transfer reaction of the well-known enzyme human carbonic anhydrase II. The potential of mean force, F( r), in the absence of the chemical step is found to reproduce earlier results on the equilibrium population of two side-chain orientations of key residue His-64. Estimation of rate constants, k, from mean first passage times for the three different stages of catalysis shows that the rate-determining step of intramolecular proton transfer occurs with k ≃ 1.0 × 10 6 s -1 , in close agreement with known experimental results.

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. Copyright © 2015. Published by Elsevier Inc.

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.

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.

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase.

Directory of Open Access Journals (Sweden)

Christina Schueler

Full Text Available Transport metabolons have been discussed between carbonic anhydrase II (CAII and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1, to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM, while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.

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 pK a 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. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Directory of Open Access Journals (Sweden)

Mayank Aggarwal

2014-03-01

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

Phylogenomic Analysis and Dynamic Evolution of Chloroplast Genomes in Salicaceae

Directory of Open Access Journals (Sweden)

Yuan Huang

2017-06-01

Full Text Available Chloroplast genomes of plants are highly conserved in both gene order and gene content. Analysis of the whole chloroplast genome is known to provide much more informative DNA sites and thus generates high resolution for plant phylogenies. Here, we report the complete chloroplast genomes of three Salix species in family Salicaceae. Phylogeny of Salicaceae inferred from complete chloroplast genomes is generally consistent with previous studies but resolved with higher statistical support. Incongruences of phylogeny, however, are observed in genus Populus, which most likely results from homoplasy. By comparing three Salix chloroplast genomes with the published chloroplast genomes of other Salicaceae species, we demonstrate that the synteny and length of chloroplast genomes in Salicaceae are highly conserved but experienced dynamic evolution among species. We identify seven positively selected chloroplast genes in Salicaceae, which might be related to the adaptive evolution of Salicaceae species. Comparative chloroplast genome analysis within the family also indicates that some chloroplast genes are lost or became pseudogenes, infer that the chloroplast genes horizontally transferred to the nucleus genome. Based on the complete nucleus genome sequences from two Salicaceae species, we remarkably identify that the entire chloroplast genome is indeed transferred and integrated to the nucleus genome in the individual of the reference genome of P. trichocarpa at least once. This observation, along with presence of the large nuclear plastid DNA (NUPTs and NUPTs-containing multiple chloroplast genes in their original order in the chloroplast genome, favors the DNA-mediated hypothesis of organelle to nucleus DNA transfer. Overall, the phylogenomic analysis using chloroplast complete genomes clearly elucidates the phylogeny of Salicaceae. The identification of positively selected chloroplast genes and dynamic chloroplast-to-nucleus gene transfers in

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

International Nuclear Information System (INIS)

Lewis, S.E.; Barnett, L.B.; Erickson, R.P.; Venta, P.J.; Tashian, R.E.

1988-01-01

Electrophoretic screening of (C57BL/6J x DBA/2J)F 1 progeny of male mice treated with N-ethyl-N-nitrosourea revealed a mouse that lacked the paternal carbonic anhydrase II (Ca II). Breeding tests showed that this trait was heritable and due to a null mutation at the Car-2 locus on chromosome 3. Like humans with the same inherited enzyme defect, animals homozygous for the new null allele are runted and have renal tubular acidosis. However, the prominent osteopetrosis found in humans with CA II deficiency could be detected even in very old homozygous null mice. A molecular analysis of the deficient mice shows that the mutant gene is not deleted and is transcribed. The CA II protein, which is normally expressed in most tissues, could not be detected by immunodiffusion analysis in any tissues of the CA II-deficient mice, suggesting a nonsense or a missense mutation at the Car-2 locus

1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads.

Science.gov (United States)

Ferraroni, Marta; Lucarini, Laura; Masini, Emanuela; Korsakov, Mikhail; Scozzafava, Andrea; Supuran, Claudiu T; Krasavin, Mikhail

2017-09-01

Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synthesized on multigram scale, which enables further development. These compounds were found to be comparable in efficacy to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for unusually high potency of these compounds became understood from their high-resolution X-ray crystallography structures. These data significantly expand our understanding of heterocycle-based primary sulfonamides, many of which have recently emerged from our labs - particularly, from the corneal permeability standpoint. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis

Directory of Open Access Journals (Sweden)

Qian Zhao

2018-01-01

Full Text Available Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and β subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead

Utilization of complete chloroplast genomes for phylogenetic studies

NARCIS (Netherlands)

Ramlee, Shairul Izan Binti

2016-01-01

Chloroplast DNA sequence polymorphisms are a primary source of data in many plant phylogenetic studies. The chloroplast genome is relatively conserved in its evolution making it an ideal molecule to retain phylogenetic signals. The chloroplast genome is also largely, but not completely, free from

Photosynthesis by isolated chloroplasts. IV. General concept and comparison of three photochemical reactions

Energy Technology Data Exchange (ETDEWEB)

Arnon, D I; Allen, M B; Whatley, F R

1956-01-01

Procedures are described for the preparation of chloroplasts capable of carrying out three photochemical reactions, each representing an increasingly complex phase of photosynthesis: photolysis of water (Hill reaction), esterification of inorganic phosphate into adenosine triphosphate (photosynthetic phosphorylation) and the reduction of carbon dioxide to the level of carbohydrates with a simultaneous evolution of oxygen. The three photochemical reactions were separable by variations in the technique for preparation of chloroplasts and by differential inhibition by several reagents. Inhibition of a more complex phase of photosynthesis does not affect the simpler one which precedes it and, conversely, the inhibition of a simpler phase of photosynthesis is paralleled by an inhibition of the more complex phase which follows. Reversible inhibition of CO/sub 2/ fixation and photosynthetic phosphorylation, but not of photolysis, by sulfhydryl group inhibitors suggests that sulfhydryl compounds (enzymes, cofactors, or both) are involved in phosphorylation and CO/sub 2/ fixation, but not in the primary conversion of light into chemical energy as measured by the Hill reaction. Evidence is presented in support of the conclusion that the synthesis of ATP by green cells occurs at two distinct sites: anaerobically in chloroplasts by photosynthetic phosphorylation, and acrobically in smaller cytoplasmic particles, presumably mitochondria, by oxidative phosphorylation independent of light. A general scheme of photosynthesis by chloroplasts, consistent with these findings, is presented. 44 references, 8 figures, 4 tables.

Non radioactive precursor import into chloroplasts

International Nuclear Information System (INIS)

Lombardo, V.A.; Ottado, J.

2003-01-01

Full text: Eukaryotic cells have a subcellular organization based on organelles. Protein transport to these organelles is quantitatively important because the majority of cellular proteins are codified in nuclear genes and then delivered to their final destination. Most of the chloroplast proteins are translated on cytoplasmic ribosomes as larger precursors with an amino terminal transit peptide that is necessary and sufficient to direct the precursor to the chloroplast. Once inside the organelle the transit peptide is cleaved and the mature protein adopts its folded form. In this work we developed a system for the expression and purification of the pea ferredoxin-NADP + reductase precursor (preFNR) for its import into chloroplasts in non radioactive conditions. We constructed a preFNR fused in its carboxy terminus to a 6 histidines peptide (preFNR-6xHis) that allows its identification using a commercial specific antibody. The construction was expressed, purified, processed and precipitated, rendering a soluble and active preFNR-6xHis that was used in binding and import into chloroplasts experiments. The reisolated chloroplasts were analyzed by SDS-PAGE, electro-blotting and revealed by immuno-detection using either colorimetric or chemiluminescent reactive. We performed also import experiments labeling preFNR and preFNR-6xHis with radioactive methionine as controls. We conclude that preFNR-6xHis is bound and imported into chloroplasts as the wild type preFNR and that both colorimetric or chemiluminescent detection methods are useful to avoid the manipulation of radioactive material. (author)

Complex chloroplast RNA metabolism: just debugging the genetic programme?

Directory of Open Access Journals (Sweden)

Schmitz-Linneweber Christian

2008-08-01

Full Text Available Abstract Background The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity. Results We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants. Conclusion Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.

Complete chloroplast genome of Gracilaria firma (Gracilariaceae, Rhodophyta), with discussion on the use of chloroplast phylogenomics in the subclass Rhodymeniophycidae.

Science.gov (United States)

Ng, Poh-Kheng; Lin, Showe-Mei; Lim, Phaik-Eem; Liu, Li-Chia; Chen, Chien-Ming; Pai, Tun-Wen

2017-01-06

The chloroplast genome of Gracilaria firma was sequenced in view of its role as an economically important marine crop with wide industrial applications. To date, there are only 15 chloroplast genomes published for the Florideophyceae. Apart from presenting the complete chloroplast genome of G. firma, this study also assessed the utility of genome-scale data to address the phylogenetic relationships within the subclass Rhodymeniophycidae. The synteny and genome structure of the chloroplast genomes across the taxa of Eurhodophytina was also examined. The chloroplast genome of Gracilaria firma maps as a circular molecule of 187,001 bp and contains 252 genes, which are distributed on both strands and consist of 35 RNA genes (3 rRNAs, 30 tRNAs, tmRNA and a ribonuclease P RNA component) and 217 protein-coding genes, including the unidentified open reading frames. The chloroplast genome of G. firma is by far the largest reported for Gracilariaceae, featuring a unique intergenic region of about 7000 bp with discontinuous vestiges of red algal plasmid DNA sequences interspersed between the nblA and cpeB genes. This chloroplast genome shows similar gene content and order to other Florideophycean taxa. Phylogenomic analyses based on the concatenated amino acid sequences of 146 protein-coding genes confirmed the monophyly of the classes Bangiophyceae and Florideophyceae with full nodal support. Relationships within the subclass Rhodymeniophycidae in Florideophyceae received moderate to strong nodal support, and the monotypic family of Gracilariales were resolved with maximum support. Chloroplast genomes hold substantial information that can be tapped for resolving the phylogenetic relationships of difficult regions in the Rhodymeniophycidae, which are perceived to have experienced rapid radiation and thus received low nodal support, as exemplified in this study. The present study shows that chloroplast genome of G. firma could serve as a key link to the full resolution of

Chloroplast microsatellite markers for Pseudotaxus chienii developed from the whole chloroplast genome of Taxus chinensis var. mairei (Taxaceae).

Science.gov (United States)

Deng, Qi; Zhang, Hanrui; He, Yipeng; Wang, Ting; Su, Yingjuan

2017-03-01

Pseudotaxus chienii (Taxaceae) is an old rare species endemic to China that has adapted well to ecological heterogeneity with high genetic diversity in its nuclear genome. However, the genetic variation in its chloroplast genome is unknown. Eighteen chloroplast microsatellite markers (cpSSRs) were developed from the whole chloroplast genome of Taxus chinensis var. mairei and successfully amplified in four P. chienii populations and one T. chinensis var. mairei population. Of these loci, 10 were polymorphic in P. chienii , whereas six were polymorphic in T. chinensis var. mairei . The unbiased haploid diversity per locus ranged from 0.000 to 0.641 and 0.000 to 0.545 for P. chienii and T. chinensis var. mairei , respectively. The 18 cpSSRs will be used to further investigate the chloroplast genetic structure and adaptive evolution in P. chienii populations.

Chloroplasts in anther endothecium of Zea mays (Poaceae).

Science.gov (United States)

Murphy, Katherine M; Egger, Rachel L; Walbot, Virginia

2015-11-01

Although anthers of Zea mays, Oryza sativa, and Arabidopsis thaliana have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to anther anatomical and ultrastructural descriptions have been reported. For example, no transmission electron microscopy (TEM) images of the premeiotic maize anther have been published. Here we report the presence of chloroplasts in maize anthers. TEM imaging, electron acceptor photosynthesis assay, in planta photon detection, microarray analysis, and light and fluorescence microscopy were used to investigate the presence of chloroplasts in the maize anther. Most cells of the maize subepidermal endothecium have starch-containing chloroplasts that do not conduct measurable photosynthesis in vitro. The maize anther contains chloroplasts in most subepidermal, endothecial cells. Although maize anthers receive sufficient light to photosynthesize in vivo and the maize anther transcribes >96% of photosynthesis-associated genes found in the maize leaf, no photosynthetic light reaction activity was detected in vitro. The endothecial cell layer should no longer be defined as a complete circle viewed transversely in anther lobes, because chloroplasts are observed only in cells directly beneath the epidermis and not those adjacent to the connective tissue. We propose that chloroplasts be a defining characteristic of differentiated endothecial cells and that nonsubepidermal endothecial cells that lack chloroplasts be defined as a separate cell type, the interendothecium. © 2015 Botanical Society of America.

Salivary carbonic anhydrase VI and its relation to salivary flow rate and buffer capacity in pregnant and non-pregnant women.

Science.gov (United States)

Kivelä, Jyrki; Laine, Merja; Parkkila, Seppo; Rajaniemi, Hannu

2003-08-01

Previous studies have shown that pregnancy may have unfavourable effects on oral health. The pH and buffer capacity (BC) of paraffin-stimulated saliva, for example, have been found to decrease towards late pregnancy. Salivary carbonic anhydrase VI (CA VI) probably protects the teeth by accelerating the neutralization of hydrogen ions in the enamel pellicle on dental surfaces. Since estrogens and androgens are known to regulate CA expression in some tissues, we studied here whether salivary CA VI concentration shows pregnancy-related changes. Paraffin-stimulated salivary samples were collected from nine pregnant women 1 month before delivery and about 2 months afterwards and assayed for salivary CA VI concentration, BC and flow rate. The enzyme concentration was determined using a specific time-resolved immunofluorometric assay. The control group consisted of 17 healthy non-pregnant women. The results indicated that salivary CA VI levels varied markedly among individuals, but no significant differences in mean concentrations were seen between the samples collected during late pregnancy and postpartum. BC values were lower during pregnancy, however. Our findings suggest that CA VI secretion is not significantly affected by the hormonal alterations associated with pregnancy, and confirm the earlier reports that CA VI is not involved in the regulation of actual salivary BC.

The role of hypoxia response element in TGFβ-induced carbonic anhydrase IX expression in Hep3B human hepatoma cells

Directory of Open Access Journals (Sweden)

Yildirim Hatice

2017-01-01

Full Text Available Carbonic anhydrase IX (CAIX is a hypoxia-regulated gene. It is over expressed in a variety of cancers, including hepatocellular cancer. Transforming growth factor β (TGFβ is considered to have an impact on cancer biology due to its important roles in cell proliferation and differentiation. The effect of the TGFβ on CAIX expression under hypoxia and the mechanism underlying the role of the hypoxia response element (HRE on this expression are unknown. In this study, we demonstrate that TGFβ upregulates CAIX expression under hypoxic conditions in the Hep3B hepatoma cell line, indicating that the mitogen-activated protein kinase (MAPK- and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K-signaling pathways might be responsible for this response. Site-directed mutagenesis of the HRE region in CAIX promoter reduced the TGFβ-induced CAIX promoter activity, pointing to the significance of HRE for this response. Up regulation of TGFβ-stimulated CAIX expression was consistent with the up regulation of promoter activity of five different truncated constructs of the CAIX promoter under hypoxia. Our findings show that the HRE region is critical for TGFβ-induced CAIX expression, which is mainly controlled by MAPK and PI3K pathways.

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

Science.gov (United States)

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

Mechanism of protein import across the chloroplast envelope.

Science.gov (United States)

Chen, K; Chen, X; Schnell, D J

2000-01-01

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43 Is Required for Chloroplast Development and Photosynthesis.

Directory of Open Access Journals (Sweden)

Xiang-guang Lv

Full Text Available A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS-induced IR64 (Oryza sativa L. ssp. indica mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43 with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43 was required for the normal development of chloroplasts and photosynthesis in rice.

Fatty acid synthesis by spinach chloroplasts, 2

International Nuclear Information System (INIS)

Yamada, Mitsuhiro; Nakamura, Yasunori

1975-01-01

By incorporation of 3 H 2 O into the fatty acid chain in the presence of unlabelled precursor, we showed that fatty acids are synthesized from PGA, PEP and pyruvate by intact spinach chloroplasts in the light. 13 C-tracer experiments confirmed that 1-C of pyruvate is decarboxylated and 2-C is incorporated into fatty acids by the chloroplasts. The patterns of fatty acids synthesized from PGA and pyruvate were the same as that from acetate. The highest rate of fatty acid synthesis was reached at the physiological concentration of PGA (3 mM) and pyruvate (1 mM). These results indicate the operation of the following path in the chloroplasts in light: PGA→PEP→pyruvate→acetylCoA→fatty acids. Since citrate and OAA were much less active and malate and glyoxylate were inert as precursors for fatty acid synthesis, PEP or pyruvate carboxylation, citrate lyase reaction and malate synthetase reaction are not involved in the formation of acetylCoA and fatty acids. Since pyruvate was much more effective as a substrate for fatty acid synthesis than lactate, acetaldehyde or acetate, direct decarboxylation path is considered to be the primary path from pyruvate to acetylCoA. The insignificant effect of chloroplast-washing on fatty acid synthesis from PGA and pyruvate indicates that the glycolytic path from PGA to pyruvate is associated with the chloroplasts. Since pyruvate was more effectively incorporated into fatty acids than acetylCoA, it is unlikely that pyruvate decarboxylation to acetylCoA is due to mitochondria contaminating the chloroplast preparation. On the basis of measurements of 3 H 2 O incorporation in the light and dark, the activity of fatty acid synthesis in spincah leaves appears to be shared by the activities in chloroplasts (87%) and other organelles (13%). (author)

Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants.

Directory of Open Access Journals (Sweden)

Vivien eRolland

2016-02-01

Full Text Available Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM, principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM. At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ~37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92-115 amino acids, containing a cleavable chloroplast transit peptide (cTP and a membrane protein leader (MPL, was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope.

Early increase in circulating carbonic anhydrase IX during neoadjuvant treatment predicts favourable outcome in locally advanced rectal cancer

International Nuclear Information System (INIS)

Hektoen, Helga Helseth; Flatmark, Kjersti; Andersson, Yvonne; Dueland, Svein; Redalen, Kathrine Røe; Ree, Anne Hansen

2015-01-01

Locally advanced rectal cancer (LARC) comprises heterogeneous tumours with predominant hypoxic components. The hypoxia-inducible metabolic shift causes microenvironmental acidification generated by carbonic anhydrase IX (CAIX) and facilitates metastatic progression, the dominant cause of failure in LARC. Using a commercially available immunoassay, circulating CAIX was assessed in prospectively archived serial serum samples collected during combined-modality neoadjuvant treatment of LARC patients and correlated to histologic tumour response and progression-free survival (PFS). Patients who from their individual baseline level displayed serum CAIX increase above a threshold of 224 pg/ml (with 96 % specificity and 39 % sensitivity) after completion of short-course neoadjuvant chemotherapy (NACT) prior to long-course chemoradiotherapy and definitive surgery had significantly better 5-year PFS (94 %) than patients with below-threshold post-NACT versus baseline alteration (PFS rate of 56 %; p < 0.01). This particular CAIX parameter, ΔNACT, was significantly correlated with histologic ypT0–2 and ypN0 outcome (p < 0.01) and remained an independent PFS predictor in multivariate analysis wherein it was entered as continuous variable (p = 0.04). Our results indicate that low ΔNACT, i.e., a weak increase in serum CAIX level following initial neoadjuvant treatment (in this case two cycles of the Nordic FLOX regimen), might be used as risk-adapted stratification to postoperative therapy or other modes of intensification of the combined-modality protocol in LARC. ClinicalTrials.gov: NCT00278694

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast1

Science.gov (United States)

Noctor, Graham; Arisi, Ana-Carolina M.; Jouanin, Lise; Foyer, Christine H.

1998-01-01

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast. PMID:9765532

C4 photosynthetic machinery: insights from maize chloroplast proteomics

Directory of Open Access Journals (Sweden)

Qi eZhao

2013-04-01

Full Text Available C4 plants exhibit much higher CO2 assimilation rates than C3 plants. The specialized differentiation of mesophyll cell (M and bundle sheath cell (BS type chloroplasts is unique to C4 plants and improves photosynthesis efficiency. Maize (Zea mays is an important crop and model with C4 photosynthetic machinery. Current high-throughput quantitative proteomics approaches (e.g., 2DE, iTRAQ, and shotgun proteomics have been employed to investigate maize chloroplast structure and function. These proteomic studies have provided valuable information on C4 chloroplast protein components, photosynthesis, and other metabolic mechanisms underlying chloroplast biogenesis, stromal and membrane differentiation, as well as response to salinity, high/low temperature, and light stress. This review presents an overview of proteomics advances in maize chloroplast biology.

Development of Chloroplast Genomic Resources in Chinese Yam (Dioscorea polystachya

Directory of Open Access Journals (Sweden)

Junling Cao

2018-01-01

Full Text Available Chinese yam has been used both as a food and in traditional herbal medicine. Developing more effective genetic markers in this species is necessary to assess its genetic diversity and perform cultivar identification. In this study, new chloroplast genomic resources were developed using whole chloroplast genomes from six genotypes originating from different geographical locations. The Dioscorea polystachya chloroplast genome is a circular molecule consisting of two single-copy regions separated by a pair of inverted repeats. Comparative analyses of six D. polystachya chloroplast genomes revealed 141 single nucleotide polymorphisms (SNPs. Seventy simple sequence repeats (SSRs were found in the six genotypes, including 24 polymorphic SSRs. Forty-three common indels and five small inversions were detected. Phylogenetic analysis based on the complete chloroplast genome provided the best resolution among the genotypes. Our evaluation of chloroplast genome resources among these genotypes led us to consider the complete chloroplast genome sequence of D. polystachya as a source of reliable and valuable molecular markers for revealing biogeographical structure and the extent of genetic variation in wild populations and for identifying different cultivars.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

Science.gov (United States)

Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

2017-10-01

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response.

Science.gov (United States)

Myouga, Fumiyoshi; Motohashi, Reiko; Kuromori, Takashi; Nagata, Noriko; Shinozaki, Kazuo

2006-10-01

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

The evolution of blue-greens and the origins of chloroplasts

Science.gov (United States)

Schwartz, R. M.; Dayhoff, M. O.

1981-01-01

All of the available molecular data support the theory that the chloroplasts of eukaryote cells were originally free-living blue-greens. Of great interest is what the relationships are between contemporary types of blue-greens and eukaryote chloroplasts and whether the chloroplasts of the various eukaryotes are the result of one or more than one symbiosis. By combining information from phylogenetic trees based on cytochrome c6 and 2Fe-2S ferredoxin sequences, it is shown that the chloroplasts of a number of eukaryote algae as well as the protist Euglena are polyphyletic; the chloroplasts of green algae and the higher plants may be the result of a single symbiosis.

Proteochemometric Modeling of the Interaction Space of Carbonic Anhydrase and its Inhibitors: An Assessment of Structure-based and Sequence-based Descriptors.

Science.gov (United States)

Rasti, Behnam; Namazi, Mohsen; Karimi-Jafari, M H; Ghasemi, Jahan B

2017-04-01

Due to its physiological and clinical roles, carbonic anhydrase (CA) is one of the most interesting case studies. There are different classes of CAinhibitors including sulfonamides, polyamines, coumarins and dithiocarbamates (DTCs). However, many of them hardly act as a selective inhibitor against a specific isoform. Therefore, finding highly selective inhibitors for different isoforms of CA is still an ongoing project. Proteochemometrics modeling (PCM) is able to model the bioactivity of multiple compounds against different isoforms of a protein. Therefore, it would be extremely applicable when investigating the selectivity of different ligands towards different receptors. Given the facts, we applied PCM to investigate the interaction space and structural properties that lead to the selective inhibition of CA isoforms by some dithiocarbamates. Our models have provided interesting structural information that can be considered to design compounds capable of inhibiting different isoforms of CA in an improved selective manner. Validity and predictivity of the models were confirmed by both internal and external validation methods; while Y-scrambling approach was applied to assess the robustness of the models. To prove the reliability and the applicability of our findings, we showed how ligands-receptors selectivity can be affected by removing any of these critical findings from the modeling process. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

On the structure of the spinach chloroplast

NARCIS (Netherlands)

Thomas, J.B.; Bustraan, M.; Paris, C.H.

1952-01-01

The structure of spinach chloroplasts was investigated with the aid of the electron microscope. It has been established that: 1. 1. the outer membrane of the chloroplasts is composed of both proteins and lipoids. 2. 2. the stroma is also built up by these components. 3. 3. within the

Purification of chicken carbonic anhydrase isozyme-III (CA-III) and its measurement in White Leghorn chickens.

Science.gov (United States)

Nishita, Toshiho; Tomita, Yuichiro; Yorifuji, Daisuke; Orito, Kensuke; Ochiai, Hideharu; Arishima, Kazuyosi

2011-11-26

The developmental profile of chicken carbonic anhydrase-III (CA-III) blood levels has not been previously determined or reported. We isolated CA-III from chicken muscle and investigated age-related changes in the levels of CA-III in blood. CA-III was purified from chicken muscle. The levels of CA-III in plasma and erythrocytes from 278 female chickens (aged 1-93 weeks) and 68 male chickens (aged 3-59 weeks) were determined by ELISA. The mean level of CA-III in female chicken erythrocytes (1 week old) was 4.6 μg/g of Hb, and the CA-III level did not change until 16 weeks of age. The level then increased until 63 weeks of age (11.8 μg/g of Hb), decreased to 4.7 μg/g of Hb at 73 weeks of age, and increased again until 93 weeks of age (8.6 μg/g of Hb). The mean level of CA-III in erythrocytes from male chickens (3 weeks old) was 2.4 μg/g of Hb, and this level remained steady until 59 weeks of age. The mean plasma level of CA-III in 1-week-old female chickens was 60 ng/mL, and this level was increased at 3 weeks of age (141 ng/mL) and then remained steady until 80 weeks of age (122 ng/mL). The mean plasma level of CA-III in 3-week-old male chickens was 58 ng/mL, and this level remained steady until 59 weeks of age. We observed both developmental changes and sex differences in CA-III concentrations in White Leghorn (WL) chicken erythrocytes and plasma. Simple linear regression analysis showed a significant association between the erythrocyte CA-III level and egg-laying rate in WL-chickens 16-63 weeks of age (p < 0.01).

Protein methylation reactions in intact pea chloroplasts

International Nuclear Information System (INIS)

Niemi, K.J.

1989-01-01

Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

New bioactive silver(I) complexes: Synthesis, characterization, anticancer, antibacterial and anticarbonic anhydrase II activities

Science.gov (United States)

Ozdemir, Ummuhan O.; Ozbek, Neslihan; Genc, Zuhal Karagoz; İlbiz, Firdevs; Gündüzalp, Ayla Balaban

2017-06-01

Silver(I) complexes of alkyl sulfonic acide hydrazides were newly synthesized as homologous series. Methanesulfonic acide hydrazide (L1), ethanesulfonic acide hydrazide (L2), propanesulfonic acide hydrazide (L3) and butanesulfonic acide hydrazide (L4) were used for complexation with Ag(I) ions. The silver complexes obtained in the mol ratio of 1:2 have the structural formula as Ag(L1)2NO3 (I), Ag(L2)2NO3 (II), Ag(L3)2NO3(III), (Ag(L4)2NO3 (IV). The Ag(I) complexes exhibit distorted linear two-fold coordination in [AgL2]+ cations with uncoordinated nitrates. Ligands are chelated with silver(I) ions through unsubstituted primary nitrogen in hydrazide group. Ag(I) complexes were characterized by using elemental analysis, spectroscopic methods (FT-IR, LC-MS), magnetic susceptibility and conductivity measurements. Silver(I) complexes were optimized using PBEPBE/LanL2DZ/DEF2SV basic set performed by DFT method with the Gaussian 09 program package. The geometrical parameters, frontier molecular orbitals (HOMOs and LUMOs) and molecular electrostatic potential (MEP) mapped surfaces of the optimized geometries were also determined by this quantum set. The anticancer activities of silver(I) complexes on MCF-7 human breast cancer cell line were investigated by comparing IC50 values. The antibacterial activities of complexes were studied against Gram positive bacteria; S. aureus ATCC 6538, B. subtilis ATCC 6633, B. cereus NRRL-B-3711, E. faecalis ATCC 29212 and Gram negative bacteria; E. coli ATCC 11230, P. aeruginosa ATCC 15442, K. pneumonia ATCC 70063 by using disc diffusion method. The inhibition activities of Ag(I) complexes on carbonic anhydrase II enzyme (hCA II) were also investigated by comparing IC50 and Ki values. The biological activity screening shows that Ag(I) complex of butanesulfonicacidehydrazide (IV) has the highest activity against tested breast cancer cell lines MCF-7, Gram positive/Gram negative bacteria and carbonic anhydrase II (hCA II) isoenzyme.

Novel genetic markers of the carbonic anhydrase II gene associated with egg production and reproduction traits in Tsaiya ducks.

Science.gov (United States)

Chang, M-T; Cheng, Y-S; Huang, M-C

2013-02-01

In our previous cDNA microarray study, we found that the carbonic anhydrase II (CA2) gene is one of the differentially expressed transcripts in the duck isthmus epithelium during egg formation period. The aim of this study was to identify the single-nucleotide polymorphisms (SNPs) in the CA2 gene of Tsaiya ducks. The relationship of SNP genotype with egg production and reproduction traits was also investigated. A total of 317 ducks from two lines, a control line with no selection and a selected line, were employed for testing. Three SNPs (C37T, A62G and A65G) in the 3'-untranslated region of the CA2 gene were found. SNP-trait association analysis showed that SNP C37T and A62G were associated with duck egg weight besides fertility. The ducks with the CT and AG genotypes had a 1.46 and 1.62 g/egg lower egg weight as compared with ducks with the CC and AA genotypes, respectively (p ducks with CT and AG genotypes had 5.20% and 4.22% higher fertility than those with CC and AA genotypes, respectively (p duck fertility, and the diplotype H1H4 was dominant for duck fertility. These findings might provide the basis for balanced selection and may be used in marker-assisted selection to improve egg weight and fertility simultaneously in the Tsaiya ducks. © 2012 Blackwell Verlag GmbH.

Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

Science.gov (United States)

Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

2018-12-01

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

Carbonic Anhydrase and Urease Inhibitory Potential of Various Plant Phenolics Using in vitro and in silico Methods.

Science.gov (United States)

Rauf, Abdur; Raza, Muslim; Saleem, Muhammad; Ozgen, Ufuk; Karaoglan, Esen Sezen; Renda, Gulin; Palaska, Erhan; Orhan, Ilkay Erdogan

2017-06-01

Plant phenolics are known to display many pharmacological activities. In the current study, eight phenolic compounds, e.g., luteolin 5-O-β-glucoside (1), methyl rosmarinate (2), apigenin (3), vicenin 2 (4), lithospermic acid (5), soyasaponin II (6), rubiadin 3-O-β-primeveroside (7), and 4-(β-d-glucopyranosyloxy)benzyl 3,4-dihydroxybenzoate (8), isolated from various plant species were tested at 0.2 mm against carbonic anhydrase-II (CA-II) and urease using microtiter assays. Urease inhibition rate for compounds 1 - 8 ranged between 5.0 - 41.7%, while only compounds 1, 2, and 4 showed a considerable inhibition over 50% against CA-II with the IC 50 values of 73.5 ± 1.05, 39.5 ± 1.14, and 104.5 ± 2.50 μm, respectively, where IC 50 of the reference (acetazolamide) was 21.0 ± 0.12 μm. In silico experiments were also performed through two docking softwares (Autodock Vina and i-GEMDOCK) in order to find out interactions between the compounds and CA-II. Actually, compounds 6 (30.0%) and 7 (42.0%) possessed a better binding capability toward the active site of CA-II. According to our results obtained in this study, among the phenolic compounds screened, particularly 1, 2, and 4 appear to be the promising inhibitors of CA-II and may be further investigated as possible leads for diuretic, anti-glaucoma, and antiepileptic agents. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

A protocol for expression of foreign genes in chloroplasts.

Science.gov (United States)

Verma, Dheeraj; Samson, Nalapalli P; Koya, Vijay; Daniell, Henry

2008-01-01

Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in approximately 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.

The complete chloroplast genome of the Dendrobium strongylanthum (Orchidaceae: Epidendroideae).

Science.gov (United States)

Li, Jing; Chen, Chen; Wang, Zhe-Zhi

2016-07-01

Complete chloroplast genome sequence is very useful for studying the phylogenetic and evolution of species. In this study, the complete chloroplast genome of Dendrobium strongylanthum was constructed from whole-genome Illumina sequencing data. The chloroplast genome is 153 058 bp in length with 37.6% GC content and consists of two inverted repeats (IRs) of 26 316 bp. The IR regions are separated by large single-copy region (LSC, 85 836 bp) and small single-copy (SSC, 14 590 bp) region. A total of 130 chloroplast genes were successfully annotated, including 84 protein coding genes, 38 tRNA genes, and eight rRNA genes. Phylogenetic analyses showed that the chloroplast genome of Dendrobium strongylanthum is related to that of the Dendrobium officinal.

Inorganic Carbon Utilization of the Freshwater Red Alga Compsopogon coeruleus (Balbis Montagne (Compsopogonaceae, Rhodophyta Evaluated by in situ Measurement of Chlorophyll Fluorescence

Directory of Open Access Journals (Sweden)

Shao-Lun Liu

2004-09-01

Full Text Available To explore the inorganic carbon utilization of the freshwater red alga Compsopogon coeruleus, photosynthetic rates in response to increasing of bicarbonate concentration, the addition of alkaline HEPES buffer (pH 8.8, acid HEPES buffer (pH 4.0 and the extracellular carbonic anhydrase inhibitor (acetazolamide, AZ, respectively, were examined in situ by using a submersible pulse amplitude modulated (PAM fluorometer. Among the treatments, adding acid HEPES buffer significantly reduced photosynthetic rates of the alga, while others showed no effect. Accordingly, we concluded that C. coeruleus had less or no inorganic carbon (Ci limitation in its natural habitat. The alga might have higher affinity for bicarbonate and directly uptake bicarbonate as main Ci source without the aid of extracellular carbonic anhydrase.

De Novo Assembly of Complete Chloroplast Genomes from Non-model Species Based on a K-mer Frequency-Based Selection of Chloroplast Reads from total DNA Sequences.

NARCIS (Netherlands)

Izan, Shairul; Esselink, G.; Visser, R.G.F.; Smulders, M.J.M.; Borm, T.J.A.

2017-01-01

Whole Genome Shotgun (WGS) sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This

Oxidative damage to chloroplasts from Chlorella vulgaris exposed to ultraviolet-B radiation

International Nuclear Information System (INIS)

Malanga, G.; Calmanovici, G.; Puntarulo, S.

1997-01-01

Upon UV-B irradiation, Chlorella vulgaris cells and isolated chloroplasts increased in size and starch accumulation. Photosynthetic capacity and chlorophyll content of chloroplasts isolated from irradiated algae decreased by 72 and 66%, as compared to chloroplasts isolated from control cells. Dihydrorhodamine 123 conversion to rhodamine 123 was used as a sensitive method for detection of peroxide (presumably hydrogen peroxide) formation in isolated chloroplasts. The accumulation of rhodamine 123 is higher in irradiated than in nonirradiated chloroplasts and the increased accumulation of rhodamine 123 depended on the UV-B dose. Quantitation of alkyl radical-EPR signals in chloroplasts indicated that UV-B exposure significantly increased radical content in the membranes. The content of an oxidized DNA base (8-hydroxy-2′-deoxyguanosine) in chloroplasts was increased by 72 and 175% after irradiation of the algal culture with 17.3 and 42.6 kJ m −2 , respectively. The chloroplastic activity of superoxide dismutase decreased by 50% as compared with control values after irradiation with 42.6 kJ m −2 and no changes in ascorbate peroxidase activity and ascorbic acid content were detected at the irradiation doses tested. The β-carotene content in chloroplasts was not affected by the irradiation, but the α-tocopherol content increased approximately 4-fold after UV-B irradiation. The results suggest that oxidative damage related to UV-B exposure is responsible for alterations in chloroplasts function and integrity, and that an antioxidant response is triggered in chloroplasts through an increase in α-tocopherol content. (author)

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

Science.gov (United States)

Lamont, Matthew G; Weber, John T

2015-06-01

The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. Copyright © 2015 Elsevier B.V. All rights reserved.

Orientation of the pigment molecules in the chloroplast

NARCIS (Netherlands)

Goedheer, J.C.

1955-01-01

Dichroism, absorption anisotropy, and anomal dispersion of birefringence were measured in the big lamellate chloroplasts of Mougeotia. The results of these measurements indicate a certain orientation of the chlorophyll molecules, and to a smaller extent, of the carotenoids in the chloroplast. In

Pb-induced avoidance-like chloroplast movements in fronds of Lemna trisulca L.

Directory of Open Access Journals (Sweden)

Sławomir Samardakiewicz

Full Text Available Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed. An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb2+, 24 h in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H2O2. In addition, we detected an enhanced accumulation of endogenous H2O2 after treatment of plants with lead. Interestingly, H2O2-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H2O2 can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the

Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.

Science.gov (United States)

Majeran, Wojciech; Zybailov, Boris; Ytterberg, A Jimmy; Dunsmore, Jason; Sun, Qi; van Wijk, Klaas J

2008-09-01

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are

Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond

Energy Technology Data Exchange (ETDEWEB)

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

2013-08-01

The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

PDV2 has a dosage effect on chloroplast division in Arabidopsis.

Science.gov (United States)

Chang, Ning; Sun, Qingqing; Li, Yiqiong; Mu, Yajuan; Hu, Jinglei; Feng, Yue; Liu, Xiaomin; Gao, Hongbo

2017-03-01

PDV2 has a dosage effect on chloroplast division in Arabidopsis thaliana , but this effect may vary in different plants. Chloroplasts have to be divided as plants grow to maintain an optimized number in the cell. Chloroplasts are divided by protein complexes across the double membranes from the stroma side to the cytosolic side. PDV2 is a chloroplast division protein on the chloroplast outer membrane. It recruits the dynamin-related GTPase ARC5 to the division site. The C-terminus of PDV2 and the C-terminus of ARC6 interact in the intermembrane space, which is important for the localization of PDV2. Previously, it was shown that overexpression of PDV2 can increase the division of chloroplasts in Arabidopsis and moss, so the authors concluded that PDV2 determines the rate of chloroplast division in land plants. PDV2 was also shown to inhibit the GTPase activity of ARC5 by in vitro experiment. These results look to be contradictory. Here, we identified a null allele of PDV2 in Arabidopsis and studied plants with different levels of PDV2. Our results suggested that the chloroplast division phenotype in Arabidopsis is sensitive to the level of PDV2, while this is not the case for ARC6. The level of PDV2 protein is reduced sharply in fast-growing leaves, while the level of ARC6 is not. The levels of PDV2 and ARC6 in several other plant species at different developmental stages were also investigated. The results indicated that their expression pattern varies in different species. Thus, PDV2 is an important positive factor of chloroplast division with an apparent dosage effect in Arabidopsis, but this effect for different chloroplast division proteins in different plants may vary.

Genetics Home Reference: carbonic anhydrase VA deficiency

Science.gov (United States)

... Salvarinova R, Yaplito-Lee J, Santra S, Shyr C, Horvath GA, Eydoux P, Lehman AM, Bernard V, Newlove T, Ukpeh H, Chakrapani A, Preece MA, Ball S, Pitt J, Vallance HD, Coulter-Mackie M, Nguyen H, Zhang LH, Bhavsar AP, Sinclair G, Waheed A, Wasserman WW, Stockler-Ipsiroglu S. Mitochondrial carbonic ...

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

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

Directory of Open Access Journals (Sweden)

Pawel Sadlecki

2014-01-01

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

Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation[OPEN

Science.gov (United States)

2018-01-01

Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization, and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental, and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems. PMID:29610211

Fluoride Alters Serum Elemental (Calcium, Magnesium, Copper, and Zinc) Homeostasis Along with Erythrocyte Carbonic Anhydrase Activity in Fluorosis Endemic Villages and Restores on Supply of Safe Drinking Water in School-Going Children of Nalgonda District, India.

Science.gov (United States)

Khandare, Arjun L; Validandi, Vakdevi; Boiroju, Naveen

2018-02-17

The present study aimed to determine the serum trace elements (copper (Cu), zinc (Zn), calcium (Ca), magnesium (Mg)) along with erythrocyte carbonic anhydrase (CA) activity and effect of intervention with safe drinking water for 5 years in the school children of fluorosis endemic area. For this purpose, three categories of villages were selected based on drinking water fluoride (F): Category I (control, F = 1.68 mg/L), category II (affected F = 3.77 mg/L), and category III (intervention village) where initial drinking water F was 4.51 mg/L, and since the last 5 years, they were drinking water containing water for 5 years in school-going children.

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

Energy Technology Data Exchange (ETDEWEB)

Doyen, Jérome [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Parks, Scott K. [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Marcié, Serge [Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Pouysségur, Jacques [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Centre Scientifique de Monaco (Monaco); Chiche, Johanna, E-mail: chiche@unice.fr [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France)

2013-01-07

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 (pH{sub i})-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 pH{sub o} manipulations and hypoxia (1% O{sub 2}) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH{sub i}-regulating capacity of fibroblasts through inhibition of Na{sup +}/H{sup +} exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH{sub i}-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 pH{sub i} 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 pH{sub i}-regulating CAs as an anti-cancer strategy.

Mergers and acquisitions: malaria and the great chloroplast heist.

Science.gov (United States)

McFadden, G I

2000-01-01

The origin of the relict chloroplast recently identified in malarial parasites has been mysterious. Several new papers suggest that the parasites obtained their chloroplasts in an ancient endosymbiotic event that also created some major algal groups.

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. Copyright © 2012 S. Karger AG, Basel.

Tumor microenvironmental changes induced by the sulfamate carbonic anhydrase IX inhibitor S4 in a laryngeal tumor model.

Directory of Open Access Journals (Sweden)

Tineke W H Meijer

Full Text Available BACKGROUND AND PURPOSE: Carbonic anhydrase IX (CAIX plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3'(3",5"-dimethylphenyl-ureidophenyl sulfamate (S4 on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding. METHODS: SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR. RESULTS: CAIX ectodomain shedding decreased after treatment with S4 (p<0.01. S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed. CONCLUSION: As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.

The in vitro effects of some pesticides on carbonic anhydrase activity of Oncorhynchus mykiss and Cyprinus carpio carpio fish.

Science.gov (United States)

Doğan, Serap

2006-05-20

Systemic carbonic anhydrase (CA) inhibitors are among the most powerful agents to lower intraocular pressure. Unfortunately, their use is frequently accompanied by undesired side effects. Some are due to the relatively large amounts of drug that have to be systematically administered to inhibit the CA in the ciliary processes. The aim of the present work was to study in vitro effects of some pesticides on CA enzyme obtained from blood of fish, which play a key role in salt- and osmoregulation and acid-base balance in the fish, Oncorhynchus mykiss and Cyprinus carpio carpio living in freshwaters, and compared with CA inhibitors. CA activities were significantly inhibited by pesticides and inhibitors. I(50) values of O. mykiss CA enzyme inhibited by lambda-cyhalothrin, deltametrin, diozinon, dorzolamide and brinzolamide were 6.05 x 10(-4), 1.48 x 10(-5), 6.84 x 10(-3), 3.82 x 10(-5) and 1.80 x 10(-6) mol/l, and that for C. c. carpio 6.86 x 10(-4), 4.70 x 10(-4), 3.92 x 10(-3), 8.34 x 10(-6) and 1.42 x 10(-6) mol/l, respectively. The pesticides used in this study inhibited the CA activity from different fish species to various degrees. It was found that the most effective inhibitor of CA enzyme within pesticides used was detrametrin. These findings observed in vitro could be useful in the understanding of the toxic effects that pesticides elicit on aquatic organisms in vivo.

The chloroplast genome of a symbiodinium sp. clade C3 isolate

KAUST Repository

Barbrook, Adrian C.

2014-01-01

Dinoflagellate algae of the genus Symbiodinium form important symbioses within corals and other benthic marine animals. Dinoflagellates possess an extremely reduced plastid genome relative to those examined in plants and other algae. In dinoflagellates the plastid genes are located on small plasmids, commonly referred to as \\'minicircles\\'. However, the chloroplast genomes of dinoflagellates have only been extensively characterised from a handful of species. There is also evidence of considerable variation in the chloroplast genome organisation across those species that have been examined. We therefore characterised the chloroplast genome from an environmental coral isolate, in this case containing a symbiont belonging to the Symbiodinium sp. clade C3. The gene content of the genome is well conserved with respect to previously characterised genomes. However, unlike previously characterised dinoflagellate chloroplast genomes we did not identify any \\'empty\\' minicircles. The sequences of this chloroplast genome show a high rate of evolution relative to other algal species. Particularly notable was a surprisingly high level of sequence divergence within the core polypeptides of photosystem I, the reasons for which are currently unknown. This chloroplast genome also possesses distinctive codon usage and GC content. These features suggest that chloroplast genomes in Symbiodinium are highly plastic. © 2013 Adrian C. Barbrook.

The chloroplast genome of a symbiodinium sp. clade C3 isolate

KAUST Repository

Barbrook, Adrian C.; Voolstra, Christian R.; Howe, Christopher J.

2014-01-01

Dinoflagellate algae of the genus Symbiodinium form important symbioses within corals and other benthic marine animals. Dinoflagellates possess an extremely reduced plastid genome relative to those examined in plants and other algae. In dinoflagellates the plastid genes are located on small plasmids, commonly referred to as 'minicircles'. However, the chloroplast genomes of dinoflagellates have only been extensively characterised from a handful of species. There is also evidence of considerable variation in the chloroplast genome organisation across those species that have been examined. We therefore characterised the chloroplast genome from an environmental coral isolate, in this case containing a symbiont belonging to the Symbiodinium sp. clade C3. The gene content of the genome is well conserved with respect to previously characterised genomes. However, unlike previously characterised dinoflagellate chloroplast genomes we did not identify any 'empty' minicircles. The sequences of this chloroplast genome show a high rate of evolution relative to other algal species. Particularly notable was a surprisingly high level of sequence divergence within the core polypeptides of photosystem I, the reasons for which are currently unknown. This chloroplast genome also possesses distinctive codon usage and GC content. These features suggest that chloroplast genomes in Symbiodinium are highly plastic. © 2013 Adrian C. Barbrook.

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

Science.gov (United States)

Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cadmium Disrupts Subcellular Organelles, Including Chloroplasts, Resulting in Melatonin Induction in Plants

Directory of Open Access Journals (Sweden)

Hyoung-Yool Lee

2017-10-01

Full Text Available Cadmium is a well-known elicitor of melatonin synthesis in plants, including rice. However, the mechanisms by which cadmium induces melatonin induction remain elusive. To investigate whether cadmium influences physical integrities in subcellular organelles, we treated tobacco leaves with either CdCl2 or AlCl3 and monitored the structures of subcellular organelles—such as chloroplasts, mitochondria, and the endoplasmic reticulum (ER—using confocal microscopic analysis. Unlike AlCl3 treatment, CdCl2 (0.5 mM treatment significantly disrupted chloroplasts, mitochondria, and ER. In theory, the disruption of chloroplasts enabled chloroplast-expressed serotonin N-acetyltransferase (SNAT to encounter serotonin in the cytoplasm, leading to the synthesis of N-acetylserotonin followed by melatonin synthesis. In fact, the disruption of chloroplasts by cadmium, not by aluminum, gave rise to a huge induction of melatonin in rice leaves, which suggests that cadmium-treated chloroplast disruption plays an important role in inducing melatonin in plants by removing physical barriers, such as chloroplast double membranes, allowing SNAT to gain access to the serotonin substrate enriched in the cytoplasm.

De Novo Assembly of Complete Chloroplast Genomes from Non-model Species Based on a K-mer Frequency-Based Selection of Chloroplast Reads from Total DNA Sequences

Directory of Open Access Journals (Sweden)

Shairul Izan

2017-08-01

Full Text Available Whole Genome Shotgun (WGS sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This re-sequencing approach may select against structural differences between the genomes especially in non-model species for which no close relatives have been sequenced before. The alternative approach is to de novo assemble the chloroplast genome from total genomic DNA sequences. In this study, we used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. Our strategy includes steps aimed at optimizing assemblies and filling gaps which are left due to coverage variation in the WGS dataset. We have successfully de novo assembled three complete chloroplast genomes from plant species with a range of nuclear genome sizes to demonstrate the universality of our approach: Solanum lycopersicum (0.9 Gb, Aegilops tauschii (4 Gb and Paphiopedilum henryanum (25 Gb. We also highlight the need to optimize the choice of k and the amount of data used. This new and cost-effective method for de novo short read assembly will facilitate the study of complete chloroplast genomes with more accurate analyses and inferences, especially in non-model plant genomes.

Complete sequencing of five araliaceae chloroplast genomes and the phylogenetic implications.

Directory of Open Access Journals (Sweden)

Rong Li

Full Text Available BACKGROUND: The ginseng family (Araliaceae includes a number of economically important plant species. Previously phylogenetic studies circumscribed three major clades within the core ginseng plant family, yet the internal relationships of each major group have been poorly resolved perhaps due to rapid radiation of these lineages. Recent studies have shown that phyogenomics based on chloroplast genomes provides a viable way to resolve complex relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report the complete nucleotide sequences of five Araliaceae chloroplast genomes using next-generation sequencing technology. The five chloroplast genomes are 156,333-156,459 bp in length including a pair of inverted repeats (25,551-26,108 bp separated by the large single-copy (86,028-86,566 bp and small single-copy (18,021-19,117 bp regions. Each chloroplast genome contains the same 114 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 80 protein coding genes. Gene size, content, and order, AT content, and IR/SC boundary structure are similar among all Araliaceae chloroplast genomes. A total of 140 repeats were identified in the five chloroplast genomes with palindromic repeat as the most common type. Phylogenomic analyses using parsimony, likelihood, and Bayesian inference based on the complete chloroplast genomes strongly supported the monophyly of the Asian Palmate group and the Aralia-Panax group. Furthermore, the relationships among the sampled taxa within the Asian Palmate group were well resolved. Twenty-six DNA markers with the percentage of variable sites higher than 5% were identified, which may be useful for phylogenetic studies of Araliaceae. CONCLUSION: The chloroplast genomes of Araliaceae are highly conserved in all aspects of genome features. The large-scale phylogenomic data based on the complete chloroplast DNA sequences is shown to be effective for the phylogenetic reconstruction of Araliaceae.

The complete chloroplast genome sequence of Helwingia himalaica (Helwingiaceae, Aquifoliales) and a chloroplast phylogenomic analysis of the Campanulidae

OpenAIRE

Yao, Xin; Liu, Ying-Ying; Tan, Yun-Hong; Song, Yu; Corlett, Richard T.

2016-01-01

Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for Helwingia himalaica, the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large (c. 35,000 species) subclass Campanulidae in order to inves...

A comparison of rice chloroplast genomes

DEFF Research Database (Denmark)

Tang, Jiabin; Xia, Hong'ai; Cao, Mengliang

2004-01-01

Using high quality sequence reads extracted from our whole genome shotgun repository, we assembled two chloroplast genome sequences from two rice (Oryza sativa) varieties, one from 93-11 (a typical indica variety) and the other from PA64S (an indica-like variety with maternal origin of japonica......), which are both parental varieties of the super-hybrid rice, LYP9. Based on the patterns of high sequence coverage, we partitioned chloroplast sequence variations into two classes, intravarietal and intersubspecific polymorphisms. Intravarietal polymorphisms refer to variations within 93-11 or PA64S...

The TOC complex: preprotein gateway to the chloroplast.

Science.gov (United States)

Andrès, Charles; Agne, Birgit; Kessler, Felix

2010-06-01

Photosynthetic eukaryotes strongly depend on chloroplast metabolic pathways. Most if not all involve nuclear encoded proteins. These are synthesized as cytosolic preproteins with N-terminal, cleavable targeting sequences (transit peptide). Preproteins are imported by a major pathway composed of two proteins complexes: TOC and TIC (Translocon of the Outer and Inner membranes of the Chloroplasts, respectively). These selectively recognize the preproteins and facilitate their transport across the chloroplast envelope. The TOC core complex consists of three types of components, each belonging to a small family: Toc34, Toc75 and Toc159. Toc34 and Toc159 isoforms represent a subfamily of the GTPase superfamily. The members of the Toc34 and Toc159 subfamily act as GTP-dependent receptors at the chloroplast surface and distinct members of each occur in defined, substrate-specific TOC complexes. Toc75, a member of the Omp85 family, is conserved from prokaryotes and functions as the unique protein-conducting channel at the outer membrane. In this review we will describe the current state of knowledge regarding the composition and function of the TOC complex.

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.

Science.gov (United States)

de Araujo, Charlotte; Arefeen, Dewan; Tadesse, Yohannes; Long, Benedict M; Price, G Dean; Rowlett, Roger S; Kimber, Matthew S; Espie, George S

2014-09-01

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.

Purification of chicken carbonic anhydrase isozyme-III (CA-III and its measurement in White Leghorn chickens

Directory of Open Access Journals (Sweden)

Nishita Toshiho

2011-11-01

Full Text Available Abstract Background The developmental profile of chicken carbonic anhydrase-III (CA-III blood levels has not been previously determined or reported. We isolated CA-III from chicken muscle and investigated age-related changes in the levels of CA-III in blood. Methods CA-III was purified from chicken muscle. The levels of CA-III in plasma and erythrocytes from 278 female chickens (aged 1-93 weeks and 68 male chickens (aged 3-59 weeks were determined by ELISA. Results The mean level of CA-III in female chicken erythrocytes (1 week old was 4.6 μg/g of Hb, and the CA-III level did not change until 16 weeks of age. The level then increased until 63 weeks of age (11.8 μg/g of Hb, decreased to 4.7 μg/g of Hb at 73 weeks of age, and increased again until 93 weeks of age (8.6 μg/g of Hb. The mean level of CA-III in erythrocytes from male chickens (3 weeks old was 2.4 μg/g of Hb, and this level remained steady until 59 weeks of age. The mean plasma level of CA-III in 1-week-old female chickens was 60 ng/mL, and this level was increased at 3 weeks of age (141 ng/mL and then remained steady until 80 weeks of age (122 ng/mL. The mean plasma level of CA-III in 3-week-old male chickens was 58 ng/mL, and this level remained steady until 59 weeks of age. Conclusion We observed both developmental changes and sex differences in CA-III concentrations in White Leghorn (WL chicken erythrocytes and plasma. Simple linear regression analysis showed a significant association between the erythrocyte CA-III level and egg-laying rate in WL-chickens 16-63 weeks of age (p

The Relationship of Oxidation Sensitivity of Red Blood Cells and Carbonic Anhydrase Activity in Stored Human Blood: Effect of Certain Phenolic Compounds

Directory of Open Access Journals (Sweden)

Zübeyir Huyut

2016-01-01

Full Text Available It has been reported that many modifications occur with the increase of oxidative stress during storage in erythrocytes. In order to delay these negative changes, we evaluated whether the addition of substances likely to protect antioxidant capacity in stored blood would be useful. Therefore, we investigated the effects of resveratrol, tannic acid, and caffeic acid in lipid peroxidation and antioxidant capacity of erythrocytes in stored blood. Donated blood was taken into four CPD containing blood bags. One bag was used as the control, and the others were supplemented with caffeic acid (30 μg/mL, resveratrol (30 μg/mL, and tannic acid (15 μg/mL, respectively. Erythrocyte lipid peroxidation, sensitivity to oxidation, glutathione levels and carbonic anhydrase, glutathione peroxidase, and catalase activities were measured on days 0, 7, 14, 21, and 28. In the control group, erythrocyte malondialdehyde levels and sensitivity to oxidation were increased whereas glutathione, glutathione peroxidase, and catalase levels were decreased (p<0.05. Resveratrol and caffeic acid prevented malondialdehyde accumulation and preserved glutathione, glutathione peroxidase, and catalase activities in erythrocytes. We demonstrated that resveratrol, caffeic acid, and tannic acid in stored blood could decrease the sensitivity to oxidation of erythrocytes in vitro but did not exhibit such effects on CA activity.

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

International Nuclear Information System (INIS)

Dubois, Ludwig; Peeters, Sarah G.J.A.; Kuijk, Simon J.A. van; Yaromina, Ala; Lieuwes, Natasja G.; Saraya, Ruchi; Biemans, Rianne; Rami, Marouan; Parvathaneni, Nanda Kumar; Vullo, Daniela; Vooijs, Marc; Supuran, Claudiu T.; Winum, Jean-Yves

2013-01-01

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

Non-destructive estimates of soil carbonic anhydrase activity and associated soil water oxygen isotope composition

Science.gov (United States)

Jones, Sam P.; Ogée, Jérôme; Sauze, Joana; Wohl, Steven; Saavedra, Noelia; Fernández-Prado, Noelia; Maire, Juliette; Launois, Thomas; Bosc, Alexandre; Wingate, Lisa

2017-12-01

The contribution of photosynthesis and soil respiration to net land-atmosphere carbon dioxide (CO2) exchange can be estimated based on the differential influence of leaves and soils on budgets of the oxygen isotope composition (δ18O) of atmospheric CO2. To do so, the activity of carbonic anhydrases (CAs), a group of enzymes that catalyse the hydration of CO2 in soils and plants, needs to be understood. Measurements of soil CA activity typically involve the inversion of models describing the δ18O of CO2 fluxes to solve for the apparent, potentially catalysed, rate of CO2 hydration. This requires information about the δ18O of CO2 in isotopic equilibrium with soil water, typically obtained from destructive, depth-resolved sampling and extraction of soil water. In doing so, an assumption is made about the soil water pool that CO2 interacts with, which may bias estimates of CA activity if incorrect. Furthermore, this can represent a significant challenge in data collection given the potential for spatial and temporal variability in the δ18O 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 inferring the rate of CO2 hydration and the δ18O of soil water from the relationship between the δ18O of CO2 fluxes and the δ18O of CO2 at the soil surface measured at different ambient CO2 conditions. This approach was tested through laboratory incubations of air-dried soils that were re-wetted with three waters of different δ18O. Gas exchange measurements were made on these soils to estimate the rate of hydration and the δ18O of soil water, followed by soil water extraction to allow for comparison. Estimated rates of CO2 hydration were 6.8-14.6 times greater than the theoretical uncatalysed rate of hydration, indicating that CA were active in these soils. Importantly, these estimates were not significantly different among water treatments, suggesting

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

Energy Technology Data Exchange (ETDEWEB)

Angelova, Angelina [University of Arizona; Park, Sang-Hycuk [University of Arizona; Kyndt, John [Bellevue University; Fitzsimmons, Kevin [University of Arizona; Brown, Judith K [University of Arizona

2013-09-01

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.

Science.gov (United States)

Albrecht, Verónica; Ingenfeld, Anke; Apel, Klaus

2006-03-01

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco). One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.

Inhibition of chloroplast protein synthesis following light chilling of tomato

International Nuclear Information System (INIS)

Kent, J.; Ort, D.

1989-01-01

In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

Chloroplasts activity and PAP-signaling regulate programmed cell death in Arabidopsis

KAUST Repository

Bruggeman, Quentin

2016-01-09

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3’-phosphoadenosine 5’-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5’-3’ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response. © 2016 American Society of Plant Biologists. All Rights Reserved.

Frataxin Is Localized to Both the Chloroplast and Mitochondrion and Is Involved in Chloroplast Fe-S Protein Function in Arabidopsis.

Directory of Open Access Journals (Sweden)

Valeria R Turowski

Full Text Available Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR and ferredoxin (Fd, two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

Analysis of protein interactions at native chloroplast membranes by ellipsometry.

Directory of Open Access Journals (Sweden)

Verena Kriechbaumer

Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

The demise of chloroplast DNA in Arabidopsis.

Science.gov (United States)

Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

2004-09-01

Although it might be expected that chloroplast DNA (cpDNA) would be stably maintained in mature leaves, we report the surprising observation that cpDNA levels decline during plastid development in Arabidopsis thaliana (Col.) until most of the leaves contain little or no DNA long before the onset of senescence. We measured the cpDNA content in developing cotyledons, rosette leaves, and cauline leaves. The amount of cpDNA per chloroplast decreases as the chloroplasts develop, reaching undetectable levels in mature leaves. In young cauline leaves, most individual molecules of cpDNA are found in complex, branched forms. In expanded cauline leaves, cpDNA is present in smaller branched forms only at the base of the leaf and is virtually absent in the distal part of the leaf. We conclude that photosynthetic activity may persist long after the demise of the cpDNA. Copyright 2004 Springer-Verlag

Phosphorus compounds, proteins, nuclease and acid phosphatase activities in isolated spinach chloroplasts

Directory of Open Access Journals (Sweden)

E. Mikulska

2015-01-01

Full Text Available This paper deals with attempts to elaborate a simple method of spinach chloroplast isolation ensuring a high proportion of intact chloroplasts. We obtained 3 preparations of isolated chloroplasts. Several preliminary analyses of the obtained chloroplast fraction were also performed. Phosphorus compounds, total protein and the enzyme activities of RNase, DNase and GPase were determined. We found: 0,36-0,59% of RNA, 0,19-0,24% of DNA, 2,1-2,9% of phospholipids and 26-28% of protein. RNase activity was very high.

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.

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

Science.gov (United States)

Kišonaitė, Miglė; Zubrienė, Asta; Čapkauskaitė, Edita; Smirnov, Alexey; Smirnovienė, Joana; Kairys, Visvaldas; Michailovienė, Vilma; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2014-01-01

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-pyrimidinylthio)acetyl]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. PMID:25493428

Photosynthetic production of diterpenoids in chloroplasts and cyanobacteria

DEFF Research Database (Denmark)

Vavitsas, Konstantinos

Terpenoids are one of the largest classes of chemical compounds, some of them with industrial interest as nutraceuticals, biofuels, or chemical feedstocks. Diterpenoids are a large terpenoid subclass, and their chemical structure consists of a core skeleton of 20 carbon atoms. This skeleton can...... be further modified by cyclizing enzymes, and be decorated by the addition of chemical groups. Even though they are mainly plant-derived compounds, diterpenoid production in photosynthetic organisms is rather unexplored, with a few successful studies reported in the literature. In this thesis, I elaborate...... on the potential of using plant chloroplasts and cyanobacteria as biosynthetic vessels, with a focus on diterpenoid production, and on the potential direct linking of photosynthesis to drive electron-consuming enzymes, such as the monooxygenases cytochrome P450s. I subsequently present the full localization...

Gene expression and enzyme activities of carbonic anhydrase and glutaminase in rat kidneys induced by chronic systemic hypoxia

Directory of Open Access Journals (Sweden)

Andi N.K. Syarifin

2015-11-01

Full Text Available Background: Hypoxia can cause acidosis. Kidney plays an essential role in maintaining acid-base balance, which involves the activities of carbonic anhydrase (CA and glutaminase (GLS. This study is aimed to determine the expression and activities of the CA9 and GLS1 enzymes in relation to hypoxia inducible factor-1α (HIF-1α, a transcription factor protein which is a marker of hypoxia.Methods: This study was an in vivo experimental study with coupled paralel design. used 25 male Sprague-Dawley rats weighing 150-200 g. Rats were divided into 5 groups: the control group (normoxic condition and 4 treatment groups. The latter were kept in a hypoxic chamber (10% O2: 90% N2 for 1, 3, 5 and 7 days. All rats were euthanized after treatment, kidneys excised, tissues homogenized and investigated for gene expression of CA9, GLS1 and HIF-1α. On protein level, total enzymatic activities of CA and GLS and protein of HIF-1α were also investigated. Data were analyzed statistically using ANOVA for significance, and as its alternative, used Mann-Whitney and Kruskal-Wallis test.Results: Results showed that HIF-1α mRNA increased during hypoxia, but not HIF-1α protein. It seemed that acidosis occurs in kidney tissue, indicated by increased CA9 and GLS1 mRNA expression and specific activity of total CA and GLS1. Expression of CA9 and GLS1 mRNA both showed strong positive correlation with HIF-1α mRNA, but not with HIF-1α protein.Conclusion: It is suggested that during chronic systemic hypoxia, gene expression of CA9 and GLS1 and their enzyme activities were increased as a response to acidosis and related with the expression of HIF-1α mRNA.

Comparative studies on codon usage pattern of chloroplasts and ...

Indian Academy of Sciences (India)

Unknown

different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots ... As an important organelle of plants, the chloroplast has its own genomic environment and ... leading to the suggestion that the translation mechanism and patterns of codon usage in ...

High Stromal Carbonic Anhydrase IX Expression Is Associated With Decreased Survival in p16-Negative Head-and-Neck Tumors

International Nuclear Information System (INIS)

Brockton, Nigel; Dort, Joseph; Lau, Harold; Hao, Desiree; Brar, Sony; Klimowicz, Alexander; Petrillo, Stephanie; Diaz, Roman; Doll, Corinne; Magliocco, Anthony

2011-01-01

Purpose: Head-and-neck squamous cell carcinoma (HNSCC) is the fifth most common malignancy worldwide. Alcohol use and tobacco use are the most established risk factors; however, human papilloma virus (HPV) infection is a major risk factor for a subset of HNSCCs. Although HPV-positive tumors typically present at a more advanced stage at diagnosis, they are associated with a better prognosis. Tumor hypoxia confers poor prognosis and treatment failure, but direct tumor oxygen measurement is challenging. Endogenous markers of hypoxia (EMHs) have been proposed but have not replicated the prognostic utility of direct oxygen measurement. The expression of endogenous markers of hypoxia may be influenced by oxygen-independent factors, such as the HPV status of the tumor. Methods and Materials: Consecutive cases of locally advanced HNSCC, treated with a uniform regimen of combined radiotherapy and chemotherapy, were identified. Tissue microarrays were assembled from triplicate 0.6-mm cores of archived tumor tissue. HPV status was inferred from semiquantitative p16 immunostaining and directly measured by use of HPV-specific chromogenic in situ hybridization and polymerase chain reaction. Automated quantitative fluorescent immunohistochemistry was conducted to measure epithelial and stromal expression of carbonic anhydrase IX (CAIX) and glucose transporter 1 (GLUT1). Results: High stromal CAIX expression was associated with significantly reduced overall survival (p = 0.03) in patients with p16-negative tumors. Conclusions: This is the first study to use quantitative immunohistochemistry to examine endogenous markers of hypoxia stratified by tumor p16/HPV status. Assessment of CAIX expression in p16-negative HNSCC could identify patients with the least favorable prognosis and inform therapeutic strategies.

Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae.

Science.gov (United States)

Bernhardt, Nadine; Brassac, Jonathan; Kilian, Benjamin; Blattner, Frank R

2017-06-16

Triticeae, the tribe of wheat grasses, harbours the cereals barley, rye and wheat and their wild relatives. Although economically important, relationships within the tribe are still not understood. We analysed the phylogeny of chloroplast lineages among nearly all monogenomic Triticeae taxa and polyploid wheat species aiming at a deeper understanding of the tribe's evolution. We used on- and off-target reads of a target-enrichment experiment followed by Illumina sequencing. The read data was used to assemble the plastid locus ndhF for 194 individuals and the whole chloroplast genome for 183 individuals, representing 53 Triticeae species and 15 genera. We conducted Bayesian and multispecies coalescent analyses to infer relationships and estimate divergence times of the taxa. We present the most comprehensive dated Triticeae chloroplast phylogeny and review previous hypotheses in the framework of our results. Monophyly of Triticeae chloroplasts could not be confirmed, as either Bromus or Psathyrostachys captured a chloroplast from a lineage closely related to a Bromus-Triticeae ancestor. The most recent common ancestor of Triticeae occurred approximately between ten and 19 million years ago. The comparison of the chloroplast phylogeny with available nuclear data in several cases revealed incongruences indicating past hybridizations. Recent events of chloroplast capture were detected as individuals grouped apart from con-specific accessions in otherwise monopyhletic groups.

Exploring photosynthesis evolution by comparative analysis of metabolic networks between chloroplasts and photosynthetic bacteria

Directory of Open Access Journals (Sweden)

Hou Jing

2006-04-01

Full Text Available Abstract Background Chloroplasts descended from cyanobacteria and have a drastically reduced genome following an endosymbiotic event. Many genes of the ancestral cyanobacterial genome have been transferred to the plant nuclear genome by horizontal gene transfer. However, a selective set of metabolism pathways is maintained in chloroplasts using both chloroplast genome encoded and nuclear genome encoded enzymes. As an organelle specialized for carrying out photosynthesis, does the chloroplast metabolic network have properties adapted for higher efficiency of photosynthesis? We compared metabolic network properties of chloroplasts and prokaryotic photosynthetic organisms, mostly cyanobacteria, based on metabolic maps derived from genome data to identify features of chloroplast network properties that are different from cyanobacteria and to analyze possible functional significance of those features. Results The properties of the entire metabolic network and the sub-network that consists of reactions directly connected to the Calvin Cycle have been analyzed using hypergraph representation. Results showed that the whole metabolic networks in chloroplast and cyanobacteria both possess small-world network properties. Although the number of compounds and reactions in chloroplasts is less than that in cyanobacteria, the chloroplast's metabolic network has longer average path length, a larger diameter, and is Calvin Cycle -centered, indicating an overall less-dense network structure with specific and local high density areas in chloroplasts. Moreover, chloroplast metabolic network exhibits a better modular organization than cyanobacterial ones. Enzymes involved in the same metabolic processes tend to cluster into the same module in chloroplasts. Conclusion In summary, the differences in metabolic network properties may reflect the evolutionary changes during endosymbiosis that led to the improvement of the photosynthesis efficiency in higher plants. Our

Molecular Characterization of a Dual Domain Carbonic Anhydrase From the Ctenidium of the Giant Clam, Tridacna squamosa, and Its Expression Levels After Light Exposure, Cellular Localization, and Possible Role in the Uptake of Exogenous Inorganic Carbon

Directory of Open Access Journals (Sweden)

Clarissa Z. Y. Koh

2018-03-01

Full Text Available A Dual-Domain Carbonic Anhydrase (DDCA had been sequenced and characterized from the ctenidia (gills of the giant clam, Tridacna squamosa, which lives in symbiosis with zooxanthellae. DDCA was expressed predominantly in the ctenidium. The complete cDNA coding sequence of DDCA from T. squamosa comprised 1,803 bp, encoding a protein of 601 amino acids and 66.7 kDa. The deduced DDCA sequence contained two distinct α-CA domains, each with a specific catalytic site. It had a high sequence similarity with tgCA from Tridacna gigas. In T. squamosa, the DDCA was localized apically in certain epithelial cells near the base of the ctenidial filament and the epithelial cells surrounding the tertiary water channels. Due to the presence of two transmembrane regions in the DDCA, one of the Zn2+-containing active sites could be located externally and the other one inside the cell. These results denote that the ctenidial DDCA was positioned to dehydrate HCO3- to CO2 in seawater, and to hydrate the CO2 that had permeated the apical membrane back to HCO3- in the cytoplasm. During insolation, the host clam needs to increase the uptake of inorganic carbon from the ambient seawater to benefit the symbiotic zooxanthellae; only then, can the symbionts conduct photosynthesis and share the photosynthates with the host. Indeed, the transcript and protein levels of DDCA/DDCA in the ctenidium of T. squamosa increased significantly after 6 and 12 h of exposure to light, respectively, denoting that DDCA could participate in the light-enhanced uptake and assimilation of exogenous inorganic carbon.

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

Science.gov (United States)

Barton, Kiah A; Wozny, Michael R; Mathur, Neeta; Jaipargas, Erica-Ashley; Mathur, Jaideep

2018-01-29

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 ( gl2 ) and immutans ( im ), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment. © 2018. Published by The Company of Biologists Ltd.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein methylation in pea chloroplasts

International Nuclear Information System (INIS)

Niemi, K.J.; Adler, J.; Selman, B.R.

1990-01-01

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [ 3 H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [ 3 H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [ 3 H]methyl group

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis

Directory of Open Access Journals (Sweden)

RICARDO I TEJOS

2010-01-01

Full Text Available The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

Science.gov (United States)

Lian, Hong; Zeldes, Benjamin M; Lipscomb, Gina L; Hawkins, Aaron B; Han, Yejun; Loder, Andrew J; Nishiyama, Declan; Adams, Michael W W; Kelly, Robert M

2016-12-01

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 - and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO 2 into 3HP production in metabolically engineered P

The diurnal logic of the expression of the chloroplast genome in Chlamydomonas reinhardtii.

Directory of Open Access Journals (Sweden)

Adam D Idoine

Full Text Available Chloroplasts are derived from cyanobacteria and have retained a bacterial-type genome and gene expression machinery. The chloroplast genome encodes many of the core components of the photosynthetic apparatus in the thylakoid membranes. To avoid photooxidative damage and production of harmful reactive oxygen species (ROS by incompletely assembled thylakoid protein complexes, chloroplast gene expression must be tightly regulated and co-ordinated with gene expression in the nucleus. Little is known about the control of chloroplast gene expression at the genome-wide level in response to internal rhythms and external cues. To obtain a comprehensive picture of organelle transcript levels in the unicellular model alga Chlamydomonas reinhardtii in diurnal conditions, a qRT-PCR platform was developed and used to quantify 68 chloroplast, 21 mitochondrial as well as 71 nuclear transcripts in cells grown in highly controlled 12 h light/12 h dark cycles. Interestingly, in anticipation of dusk, chloroplast transcripts from genes involved in transcription reached peak levels first, followed by transcripts from genes involved in translation, and finally photosynthesis gene transcripts. This pattern matches perfectly the theoretical demands of a cell "waking up" from the night. A similar trend was observed in the nuclear transcripts. These results suggest a striking internal logic in the expression of the chloroplast genome and a previously unappreciated complexity in the regulation of chloroplast genes.

Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

International Nuclear Information System (INIS)

Gupta, Preeti; Deep, Shashank

2014-01-01

Highlights: • HCAII forms amyloid-like aggregates at moderate concentration of trifluoroethanol. • Protein adopts a state between β-sheet and α-helix at moderate % of TFE. • Hydrophobic surface(s) of partially structured conformation forms amyloid. • High % of TFE induces stable α-helical state preventing aggregation. - Abstract: In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII

The complete structure of the chloroplast 70S ribosome in complex with translation factor pY.

Science.gov (United States)

Bieri, Philipp; Leibundgut, Marc; Saurer, Martin; Boehringer, Daniel; Ban, Nenad

2017-02-15

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

14C fixation by leaves and leaf cell protoplasts of the submerged aquatic angiosperm Potamogeton lucens: Carbon dioxide or bicarbonate?

International Nuclear Information System (INIS)

Staal, M.; Elzenga, J.T.M.; Prins, H.B.A.

1989-01-01

Protoplasts were isolated from leaves of the aquatic angiosperm Potamogeton lucens L. The leaves utilize bicarbonate as a carbon source for photosynthesis, and show polarity; that is acidification of the periplasmic space of the lower, and alkalinization of the space near the upper leaf side. At present there are two models under consideration for this photosynthetic bicarbonate utilization process: conversion of bicarbonate into free carbon dioxide as a result of acidification and, second, a bicarbonate-proton symport across the plasma membrane. Carbon fixation of protoplasts was studied at different pH values and compared with that in leaf strips. Using the isotopic disequilibrium technique, it was established that carbon dioxide and not bicarbonate was the form in which DIC actually crossed the plasma membrane. It is concluded that there is probably no true bicarbonate transport system at the plasma membrane of these cells and that bicarbonate utilization in this species apparently rests on the conversion of bicarbonate into carbon dioxide. Experiments with acetazolamide, an inhibitor of periplasmic carbonic anhydrase, and direct measurements of carbonic anhydrase activity in intact leaves indicate that in this species the role of this enzyme for periplasmic conversion of bicarbonate into carbon dioxide is insignificant

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

Science.gov (United States)

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Chloroplast Iron Transport Proteins - Function and Impact on Plant Physiology.

Science.gov (United States)

López-Millán, Ana F; Duy, Daniela; Philippar, Katrin

2016-01-01

Chloroplasts originated about three billion years ago by endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. During evolution chloroplasts of higher plants established as the site for photosynthesis and thus became the basis for all life dependent on oxygen and carbohydrate supply. To fulfill this task, plastid organelles are loaded with the transition metals iron, copper, and manganese, which due to their redox properties are essential for photosynthetic electron transport. In consequence, chloroplasts for example represent the iron-richest system in plant cells. However, improvement of oxygenic photosynthesis in turn required adaptation of metal transport and homeostasis since metal-catalyzed generation of reactive oxygen species (ROS) causes oxidative damage. This is most acute in chloroplasts, where radicals and transition metals are side by side and ROS-production is a usual feature of photosynthetic electron transport. Thus, on the one hand when bound by proteins, chloroplast-intrinsic metals are a prerequisite for photoautotrophic life, but on the other hand become toxic when present in their highly reactive, radical generating, free ionic forms. In consequence, transport, storage and cofactor-assembly of metal ions in plastids have to be tightly controlled and are crucial throughout plant growth and development. In the recent years, proteins for iron transport have been isolated from chloroplast envelope membranes. Here, we discuss their putative functions and impact on cellular metal homeostasis as well as photosynthetic performance and plant metabolism. We further consider the potential of proteomic analyses to identify new players in the field.

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology.

Science.gov (United States)

Baier, Margarete; Dietz, Karl-Josef

2005-06-01

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.

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

Directory of Open Access Journals (Sweden)

Nabendu Pore

2015-06-01

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

Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

DEFF Research Database (Denmark)

Næsted, Henrik; Holm, Agnethe; Jenkins, Tom

2004-01-01

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

Science.gov (United States)

Solti, Ádám; Kovács, Krisztina; Müller, Brigitta; Vázquez, Saúl; Hamar, Éva; Pham, Hong Diep; Tóth, Brigitta; Abadía, Javier; Fodor, Ferenc

2016-12-01

Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd 2+ , Zn 2+ , and Mn 2+ ) enhanced, whereas oxoanions (NO 3 - , SO 4 2- , and BO 3 3- ) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe 2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression.

Science.gov (United States)

Puthiyaveetil, Sujith; Allen, John F

2009-06-22

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles-chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.

Dichroism in spinach chloroplasts

NARCIS (Netherlands)

Thomas, J.B.; Lierop, J.H. van; Ham, M. ten

1967-01-01

In spinach chloroplasts oriented at steel-water interfaces parallel to the light beam a distinct dichroism is measured at about 680 nm. This dichroism is minimal upon addition of sucrose up to a final concentration of 0.18 M to the medium, the dichroic ratio amounting to 1.02. It is concluded that

A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification.

Science.gov (United States)

Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Song, Xiaorui; Wang, Lingling; Song, Linsheng

2017-08-01

Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO 2 to HCO 3 - and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO 2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO 2 also caused the decrease of intracellular Ca 2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca 2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca 2+ content caused by Ca 2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO 2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of UV-B radiation on chloroplast translation in Pisum sativum

International Nuclear Information System (INIS)

Raab, M.M.; Jagendorf, A.T.

1990-01-01

UV-B radiation has previously been reported to reduce growth, flowering, and net photosynthesis. The present study examines the effect of UV-B radiation on isolated chloroplast of 7-10 day old pea seedlings. Amount of ( 3 H)-Leu incorporated into isolated chloroplasts was measured in the presence or absence of UV-B exposure. Preliminary experiments show a 30% inhibition of protein synthesis in isolated chloroplasts after only 20 mins of UV-B exposure (6.9 J/m 2 /30 min). Percent inhibition of chloroplast translation is directly correlated with UV-B exposure over a 60 min time span. Preliminary studies also show no change in both cold and radiolabeled protein profiles as expressed on 1-D PAGE and autofluorography. Comparative studies on the sensitivity of e - flow vs protein synthesis following UV-B exposure are underway. Further work on the role of oxygen free radicals and the specific site of action of UV-B damage to the translation machinery of chloroplasts will be discussed

Evaluation of 177Lu[Lu]-CHX-A″-DTPA-6A10 Fab as a radioimmunotherapy agent targeting carbonic anhydrase XII.

Science.gov (United States)

Fiedler, L; Kellner, M; Gosewisch, A; Oos, R; Böning, G; Lindner, S; Albert, N; Bartenstein, P; Reulen, H-J; Zeidler, R; Gildehaus, F J

2018-05-01

Due to their infiltrative growth behavior, gliomas have, even after surgical resection, a high recurrence tendency. The approach of intracavitary radioimmunotherapy (RIT) is aimed at inhibiting tumor re-growth by directly administering drugs into the resection cavity (RC). Direct application of the radioconjugate into the RC has the advantage of bypassing the blood-brain barrier, which allows the administration of higher radiation doses than systemic application. Carbonic anhydrase XII (CA XII) is highly expressed on glioma cells while being absent from normal brain and thus an attractive target molecule for RIT. We evaluated a CA XII-specific 6A10 Fab (fragment antigen binding) labelled with 177 Lu as an agent for RIT. 6A10 Fab fragment was modified and radiolabelled with 177 Lu and characterized by MALDI-TOF, flow cytometry and radio-TLC. In vitro stability was determined under physiological conditions. Biodistribution studies, autoradiography tumor examinations and planar scintigraphy imaging were performed on SCID-mice bearing human glioma xenografts. The in vitro CA XII binding capacity of the modified Fab was confirmed. Radiochemical purity was determined to be >90% after 72 h of incubation under physiological conditions. Autoradiography experiments proved the specific binding of the Fab to CA XII on tumor cells. Biodistribution studies revealed a tumor uptake of 3.0%ID/g after 6 h and no detectable brain uptake. The tumor-to-contralateral ratio of 10/1 was confirmed by quantitative planar scintigraphy. The radiochemical stability in combination with a successful in vivo tumor uptake shows the potential suitability for future RIT applications with the 6A10 Fab. Copyright © 2018 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

2014-01-01

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function. The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively. Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the

Regulation of chloroplast number and DNA synthesis in higher plants. Final report

Energy Technology Data Exchange (ETDEWEB)

Mullet, J.E.

1995-11-10

The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops.

Science.gov (United States)

Sharwood, Robert E

2017-01-01

494 I. 495 II. 496 III. 496 IV. 499 V. 499 VI. 501 VII. 501 VIII. 502 IX. 505 X. 506 507 References 507 SUMMARY: The uncertainty of future climate change is placing pressure on cropping systems to continue to provide stable increases in productive yields. To mitigate future climates and the increasing threats against global food security, new solutions to manipulate photosynthesis are required. This review explores the current efforts available to improve carbon assimilation within plant chloroplasts by engineering Rubisco, which catalyzes the rate-limiting step of CO 2 fixation. Fixation of CO 2 and subsequent cycling of 3-phosphoglycerate through the Calvin cycle provides the necessary carbohydrate building blocks for maintaining plant growth and yield, but has to compete with Rubisco oxygenation, which results in photorespiration that is energetically wasteful for plants. Engineering improvements in Rubisco is a complex challenge and requires an understanding of chloroplast gene regulatory pathways, and the intricate nature of Rubisco catalysis and biogenesis, to transplant more efficient forms of Rubisco into crops. In recent times, major advances in Rubisco engineering have been achieved through improvement of our knowledge of Rubisco synthesis and assembly, and identifying amino acid catalytic switches in the L-subunit responsible for improvements in catalysis. Improving the capacity of CO 2 fixation in crops such as rice will require further advances in chloroplast bioengineering and Rubisco biogenesis. © 2016 The Author. New Phytologist © 2016 New Phytologist Trust.

The complete chloroplast genome of banana (Musa acuminata, Zingiberales): insight into plastid monocotyledon evolution.

Science.gov (United States)

Martin, Guillaume; Baurens, Franc-Christophe; Cardi, Céline; Aury, Jean-Marc; D'Hont, Angélique

2013-01-01

Banana (genus Musa) is a crop of major economic importance worldwide. It is a monocotyledonous member of the Zingiberales, a sister group of the widely studied Poales. Most cultivated bananas are natural Musa inter-(sub-)specific triploid hybrids. A Musa acuminata reference nuclear genome sequence was recently produced based on sequencing of genomic DNA enriched in nucleus. The Musa acuminata chloroplast genome was assembled with chloroplast reads extracted from whole-genome-shotgun sequence data. The Musa chloroplast genome is a circular molecule of 169,972 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,338 bp) and a Small Single Copy region (SSC, 10,768 bp) separated by Inverted Repeat regions (IRs, 35,433 bp). Two forms of the chloroplast genome relative to the orientation of SSC versus LSC were found. The Musa chloroplast genome shows an extreme IR expansion at the IR/SSC boundary relative to the most common structures found in angiosperms. This expansion consists of the integration of three additional complete genes (rps15, ndhH and ycf1) and part of the ndhA gene. No such expansion has been observed in monocots so far. Simple Sequence Repeats were identified in the Musa chloroplast genome and a new set of Musa chloroplastic markers was designed. The complete sequence of M. acuminata ssp malaccensis chloroplast we reported here is the first one for the Zingiberales order. As such it provides new insight in the evolution of the chloroplast of monocotyledons. In particular, it reinforces that IR/SSC expansion has occurred independently several times within monocotyledons. The discovery of new polymorphic markers within Musa chloroplast opens new perspectives to better understand the origin of cultivated triploid bananas.

The complete chloroplast genome of banana (Musa acuminata, Zingiberales: insight into plastid monocotyledon evolution.

Directory of Open Access Journals (Sweden)

Guillaume Martin

Full Text Available Banana (genus Musa is a crop of major economic importance worldwide. It is a monocotyledonous member of the Zingiberales, a sister group of the widely studied Poales. Most cultivated bananas are natural Musa inter-(sub-specific triploid hybrids. A Musa acuminata reference nuclear genome sequence was recently produced based on sequencing of genomic DNA enriched in nucleus.The Musa acuminata chloroplast genome was assembled with chloroplast reads extracted from whole-genome-shotgun sequence data. The Musa chloroplast genome is a circular molecule of 169,972 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,338 bp and a Small Single Copy region (SSC, 10,768 bp separated by Inverted Repeat regions (IRs, 35,433 bp. Two forms of the chloroplast genome relative to the orientation of SSC versus LSC were found. The Musa chloroplast genome shows an extreme IR expansion at the IR/SSC boundary relative to the most common structures found in angiosperms. This expansion consists of the integration of three additional complete genes (rps15, ndhH and ycf1 and part of the ndhA gene. No such expansion has been observed in monocots so far. Simple Sequence Repeats were identified in the Musa chloroplast genome and a new set of Musa chloroplastic markers was designed.The complete sequence of M. acuminata ssp malaccensis chloroplast we reported here is the first one for the Zingiberales order. As such it provides new insight in the evolution of the chloroplast of monocotyledons. In particular, it reinforces that IR/SSC expansion has occurred independently several times within monocotyledons. The discovery of new polymorphic markers within Musa chloroplast opens new perspectives to better understand the origin of cultivated triploid bananas.

Inhibition of light modulation of chloroplast enzyme activity by sulfite. One of the lethal effects of SO/sub 2/

Energy Technology Data Exchange (ETDEWEB)

Anderson, L E; Duggan, J X

1977-01-01

The capacity of a particulate pea (Pisum sativum L.) leaf chloroplast system for light-modulation of enzyme activity is diminished by brief exposure to sodium sulfite and, when intact seedlings are exposed to atmospheric SO/sub 2/, the same system is inactivated. The destructive effect of this pollutant on green plants may therefore be due to disruption of the mechanism for control of carbon dioxide fixation.

Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants*

Science.gov (United States)

Mininno, Morgane; Brugière, Sabine; Pautre, Virginie; Gilgen, Annabelle; Ma, Sheng; Ferro, Myriam; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

2012-01-01

In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO2 fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO2 through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts. PMID:22547063

A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement.

Science.gov (United States)

Berg, Robert; Königer, Martina; Schjeide, Brit-Maren; Dikmak, George; Kohler, Susan; Harris, Gary C

2006-03-01

A new microcontroller-based photometric instrument for monitoring blue light dependent changes in leaf transmission (chloroplast movement) was developed based on a modification of the double-beam technique developed by Walzcak and Gabrys [(1980) Photosynthetica 14: 65-72]. A blue and red bicolor light emitting diode (LED) provided both a variable intensity blue actinic light and a low intensity red measuring beam. A phototransistor detected the intensity of the transmitted measuring light. An inexpensive microcontroller independently and precisely controlled the light emission of the bicolor LED. A typical measurement event involved turning off the blue actinic light for 100 mus to create a narrow temporal window for turning on and measuring the transmittance of the red light. The microcontroller was programmed using LogoChip Logo (http://www.wellesley.edu/Physics/Rberg/logochip/) to record fluence rate response curves. Laser scanning confocal microscopy was utilized to correlate the changes in leaf transmission with intercellular chloroplast position. In the dark, the chloroplasts in the spongy mesophyll exhibited no evident asymmetries in their distribution, however, in the palisade layer the cell surface in contact with the overlying epidermis was devoid of chloroplasts. The low light dependent decrease in leaf transmittance in dark acclimated leaves was correlated with the movement of chloroplasts within the palisade layer into the regions previously devoid of chloroplasts. Changes in leaf transmittance were evident within one minute following the onset of illumination. Minimal leaf transmittance was correlated with chloroplasts having retreated from cell surfaces perpendicular to the incident light (avoidance reaction) in both spongy and palisade layers.

Functional Disruption of a Chloroplast Pseudouridine Synthase Desensitizes Arabidopsis Plants to Phosphate Starvation

Directory of Open Access Journals (Sweden)

Shan Lu

2017-08-01

Full Text Available Phosphate (Pi deficiency is a common nutritional stress of plants in both agricultural and natural ecosystems. Plants respond to Pi starvation in the environment by triggering a suite of biochemical, physiological, and developmental changes that increase survival and growth. The key factors that determine plant sensitivity to Pi starvation, however, are unclear. In this research, we identified an Arabidopsis mutant, dps1, with greatly reduced sensitivity to Pi starvation. The dps1 phenotypes are caused by a mutation in the previously characterized SVR1 (SUPPRESSION OF VARIAGATION 1 gene, which encodes a chloroplast-localized pseudouridine synthase. The mutation of SVR1 results in defects in chloroplast rRNA biogenesis, which subsequently reduces chloroplast translation. Another mutant, rps5, which contains a mutation in the chloroplast ribosomal protein RPS5 and has reduced chloroplast translation, also displayed decreased sensitivity to Pi starvation. Furthermore, wild type plants treated with lincomycin, a chemical inhibitor of chloroplast translation, showed similar growth phenotypes and Pi starvation responses as dps1 and rps5. These results suggest that impaired chloroplast translation desensitizes plants to Pi starvation. Combined with previously published results showing that enhanced leaf photosynthesis augments plant responses to Pi starvation, we propose that the decrease in responses to Pi starvation in dps1, rps5, and lincomycin-treated plants is due to their reduced demand for Pi input from the environment.

Does Aerobic Respiration Produce Carbon Dioxide or Hydrogen Ion and Bicarbonate?

Science.gov (United States)

Swenson, Erik R

2018-05-01

Maintenance of intracellular pH is critical for clinical homeostasis. The metabolism of glucose, fatty acids, and amino acids yielding the generation of adenosine triphosphate in the mitochondria is accompanied by the production of acid in the Krebs cycle. Both the nature of this acidosis and the mechanism of its disposal have been argued by two investigators with a long-abiding interest in acid-base physiology. They offer different interpretations and views of the molecular mechanism of this intracellular pH regulation during normal metabolism. Dr. John Severinghaus has posited that hydrogen ion and bicarbonate are the direct end products in the Krebs cycle. In the late 1960s, he showed in brain and brain homogenate experiments that acetazolamide, a carbonic anhydrase inhibitor, reduces intracellular pH. This led him to conclude that hydrogen ion and bicarbonate are the end products, and the role of intracellular carbonic anhydrase is to rapidly generate diffusible carbon dioxide to minimize acidosis. Dr. Erik Swenson posits that carbon dioxide is a direct end product in the Krebs cycle, a more widely accepted view, and that acetazolamide prevents rapid intracellular bicarbonate formation, which can then codiffuse with carbon dioxide to the cell surface and there be reconverted for exit from the cell. Loss of this "facilitated diffusion of carbon dioxide" leads to intracellular acidosis as the still appreciable uncatalyzed rate of carbon dioxide hydration generates more protons. This review summarizes the available evidence and determines that resolution of this question will require more sophisticated measurements of intracellular pH with faster temporal resolution.

Effect of alkyl-N-phenylcarbamates on photochemical activity of spinach chloroplasts

International Nuclear Information System (INIS)

Sersen, F.; Kralova, K.; Macho, V.

1999-01-01

This study is aimed to investigate the effect of alkyl-N-phenylcarbamates on photosynthetic electron transport in spinach chloroplasts, to determine site of action in the photosynthetic apparatus of spinach chloroplasts and to find correlations between their structure and biological activity. (authors)

Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica

Directory of Open Access Journals (Sweden)

Qinhua Gan

2018-04-01

Full Text Available Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation. Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.

Effects of lead on enzymes of porphyrine biosynthesis in chloroplasts and erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Hampp, R.; Kriebitzsch, C.; Ziegler, H.

1974-01-01

Two enzymes of the chlorophyll biosynthesis pathway, delta-aminolevulinic acid dehydratase (ALAD) and prophobilinogenase (PBGA), show a pronounced sensitivity to lead ion, as was shown in isolated chloroplasts of spinach. It has been reported by several authors that the activity of ALAD involved in the hemoglobine-biosynthesis in erythrocytes is also very sensitive to lead ions. Spinach chloroplasts were isolated and sonicated and the enzyme activity tested. Calf blood was collected with heparin and kept at 0/sup 0/C until enzyme determination. Hemolyzed erythrocytes (rapid freezing and thawing twice) were used as the source of enzymes. The incubation mixture was the same as for chloroplasts; the hemoglobin content per test was about 44 mg (ALAD) and 91 mg (PBGA). ALAD in erythrocytes is somewhat more sensitive to lead ions than ALAD in chloroplasts. PBGA in erythrocytes is also inhibited by Pb/sup 2 +/ ions, again more than the chloroplast enzyme. At all concentrations of Pb/sup 2 +/ checked in our experiments the percentage of inhibition was higher with PBGA. 3 references, 1 figure.

Conformational changes in spinach (Spinacia oleracea leaves chloroplasts in vivo

Directory of Open Access Journals (Sweden)

Janina Godziemba-Czyż

2015-01-01

Full Text Available Changes in the surface area of chloroplasts from intact cells of spinach leaves (\tSpinacia oleracea induced by blue (370—500 nm and red (600- 850 nm light of various intensity (102 - 5x105 erg cm-1s-1 were investigated. The changes are deseribed in terms of mean surface area in , μm2 and frequency of oocurrence of surface size classes. Low intensity blue light caused enlargement of the chloroplast surface (as compared with that in darkness, whereas high intensity light markedly reduced it. Exposure of chloroplasts to red light produces an increase of the surface in proportion to the intensity of the light and irradiation time.

Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

Science.gov (United States)

Riemann, Anne; Güttler, Antje; Haupt, Verena; Wichmann, Henri; Reime, Sarah; Bache, Matthias; Vordermark, Dirk; Thews, Oliver

2018-03-05

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

Short-term effects of salt exposure on the maize chloroplast protein pattern.

Science.gov (United States)

Zörb, Christian; Herbst, Ramona; Forreiter, Christoph; Schubert, Sven

2009-09-01

It is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4 h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1e and a Ca(2+)-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem II, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na(+) on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

A database of PCR primers for the chloroplast genomes of higher plants

Science.gov (United States)

Heinze, Berthold

2007-01-01

Background Chloroplast genomes evolve slowly and many primers for PCR amplification and analysis of chloroplast sequences can be used across a wide array of genera. In some cases 'universal' primers have been designed for the purpose of working across species boundaries. However, the essential information on these primer sequences is scattered throughout the literature. Results A database is presented here which assembles published primer information for chloroplast DNA. Additional primers were designed to fill gaps where little or no primer information could be found. Amplicons are either the genes themselves (typically useful in studies of sequence variation in higher-order phylogeny) or they are spacers, introns, and intergenic regions (for studies of phylogeographic patterns within and among species). The current list of 'generic' primers consists of more than 700 sequences. Wherever possible, we give the locations of the primers in the thirteen fully sequenced chloroplast genomes (Nicotiana tabacum, Atropa belladonna, Spinacia oleracea, Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Pinus thunbergii, Marchantia polymorpha, Zea mays, Oenothera elata, Acorus calamus, Eucalyptus globulus, Medicago trunculata). Conclusion The database described here is designed to serve as a resource for researchers who are venturing into the study of poorly described chloroplast genomes, whether for large- or small-scale DNA sequencing projects, to study molecular variation or to investigate chloroplast evolution. PMID:17326828

A database of PCR primers for the chloroplast genomes of higher plants

Directory of Open Access Journals (Sweden)

Heinze Berthold

2007-02-01

Full Text Available Abstract Background Chloroplast genomes evolve slowly and many primers for PCR amplification and analysis of chloroplast sequences can be used across a wide array of genera. In some cases 'universal' primers have been designed for the purpose of working across species boundaries. However, the essential information on these primer sequences is scattered throughout the literature. Results A database is presented here which assembles published primer information for chloroplast DNA. Additional primers were designed to fill gaps where little or no primer information could be found. Amplicons are either the genes themselves (typically useful in studies of sequence variation in higher-order phylogeny or they are spacers, introns, and intergenic regions (for studies of phylogeographic patterns within and among species. The current list of 'generic' primers consists of more than 700 sequences. Wherever possible, we give the locations of the primers in the thirteen fully sequenced chloroplast genomes (Nicotiana tabacum, Atropa belladonna, Spinacia oleracea, Arabidopsis thaliana, Populus trichocarpa, Oryza sativa, Pinus thunbergii, Marchantia polymorpha, Zea mays, Oenothera elata, Acorus calamus, Eucalyptus globulus, Medicago trunculata. Conclusion The database described here is designed to serve as a resource for researchers who are venturing into the study of poorly described chloroplast genomes, whether for large- or small-scale DNA sequencing projects, to study molecular variation or to investigate chloroplast evolution.

Chloroplast Dynamics and Photosynthetic Efficiency: Final Technical Report

Energy Technology Data Exchange (ETDEWEB)

Hanson, Maureen [Cornell Univ., Ithaca, NY (United States)

2016-11-03

This project investigated the mechanism by which chloroplasts position themselves to maximize solar energy utilization, to enhance gas exchange, to minimize environmental stress, and to promote efficient exchange of metabolites with other compartments within the plant cell. Chloroplasts move within leaf cells to optimize light levels, moving toward levels of light useful for photosynthesis while moving away from excess light. Plastids sometimes extend their reach by sending out projections (stromules) that can connect anchor chloroplasts in position within the cell or provide close contacts with plasma membrane, mitochondria, peroxisomes, endoplasmic reticulum, and the nucleus. The intracellular location of chloroplasts in relation to other organelles with which they share biosynthetic pathways, such as peroxisomes and mitochondria in photorespiration, affects metabolite flow. This work contributed to the knowledge of the mechanisms of organelle movement and anchoring in specific locations in plant cells and how proteins traffic within the cell. We identified two domains on 12 of the 13 Arabidopsis myosins that were similar to the vacuole-binding (V) domain characterized in yeast and to the DIL domain characterized in yeast and mouse as required for secretory vesicle or melanosome movement, respectively. Because all of the Arabidopsis regions with homology to the V domain contain the amino acid sequence PAL, we refer to this region as the Arabidopsis PAL domain. We have used the yeast Myo2p tail structural information to model the 12 myosin XI tail domains containing the homologous PAL and DIL domains. Eight YFP::DIL domain fusions labeled peroxisomes; none labeled mitochondria or chloroplasts. Six myosin XI Vacuole domains labeled mitochondria and seven labeled Golgi bodies. The Arabidopsis myosin XI-F PAL domain and the homologous myosin XI-F PAL domain from N. benthamiana labels chloroplasts and stromules in N. benthamiana leaves. Using an Arabidopsis line

Regulation of photosynthesis and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins.

Science.gov (United States)

Perez-Martin, Alfonso; Michelazzo, Chiara; Torres-Ruiz, Jose M; Flexas, Jaume; Fernández, José E; Sebastiani, Luca; Diaz-Espejo, Antonio

2014-07-01

The hypothesis that aquaporins and carbonic anhydrase (CA) are involved in the regulation of stomatal (g s) and mesophyll (g m) conductance to CO2 was tested in a short-term water-stress and recovery experiment in 5-year-old olive plants (Olea europaea) growing outdoors. The evolution of leaf gas exchange, chlorophyll fluorescence, and plant water status, and a quantitative analysis of photosynthesis limitations, were followed during water stress and recovery. These variables were correlated with gene expression of the aquaporins OePIP1.1 and OePIP2.1, and stromal CA. At mild stress and at the beginning of the recovery period, stomatal limitations prevailed, while the decline in g m accounted for up to 60% of photosynthesis limitations under severe water stress. However, g m was restored to control values shortly after rewatering, facilitating the recovery of the photosynthetic rate. CA was downregulated during water stress and upregulated after recovery. The use of structural equation modelling allowed us to conclude that both OePIP1.1 and OePIP2.1 expression could explain most of the variations observed for g s and g m. CA expression also had a small but significant effect on g m in olive under water-stress conditions. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Protein disorder in plants: a view from the chloroplast

Directory of Open Access Journals (Sweden)

Yruela Inmaculada

2012-09-01

Full Text Available Abstract Background The intrinsically unstructured state of some proteins, observed in all living organisms, is essential for basic cellular functions. In this field the available information from plants is limited but it has been reached a point where these proteins can be comprehensively classified on the basis of disorder, function and evolution. Results Our analysis of plant genomes confirms that nuclear-encoded proteins follow the same trend than other multi-cellular eukaryotes; however, chloroplast- and mitochondria- encoded proteins conserve the patterns of Archaea and Bacteria, in agreement with their phylogenetic origin. Based on current knowledge about gene transference from the chloroplast to the nucleus, we report a strong correlation between the rate of disorder of transferred and nuclear-encoded proteins, even for polypeptides that play functional roles back in the chloroplast. We further investigate this trend by reviewing the set of chloroplast ribosomal proteins, one of the most representative transferred gene clusters, finding that the ribosomal large subunit, assembled from a majority of nuclear-encoded proteins, is clearly more unstructured than the small one, which integrates mostly plastid-encoded proteins. Conclusions Our observations suggest that the evolutionary dynamics of the plant nucleus adds disordered segments to genes alike, regardless of their origin, with the notable exception of proteins currently encoded in both genomes, probably due to functional constraints.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis

Directory of Open Access Journals (Sweden)

Zhou Xiangjun

2011-11-01

Full Text Available Abstract Background Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Results Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5 was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. Conclusions The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis).

Science.gov (United States)

Zhou, Xiangjun; Fei, Zhangjun; Thannhauser, Theodore W; Li, Li

2011-11-23

Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

Conflict amongst chloroplast DNA sequences obscures the phylogeny of a group of Asplenium ferns.

Science.gov (United States)

Shepherd, Lara D; Holland, Barbara R; Perrie, Leon R

2008-07-01

A previous study of the relationships amongst three subgroups of the Austral Asplenium ferns found conflicting signal between the two chloroplast loci investigated. Because organelle genomes like those of chloroplasts and mitochondria are thought to be non-recombining, with a single evolutionary history, we sequenced four additional chloroplast loci with the expectation that this would resolve these relationships. Instead, the conflict was only magnified. Although tree-building analyses favoured one of the three possible trees, one of the alternative trees actually had one more supporting site (six versus five) and received greater support in spectral and neighbor-net analyses. Simulations suggested that chance alone was unlikely to produce strong support for two of the possible trees and none for the third. Likelihood permutation tests indicated that the concatenated chloroplast sequence data appeared to have experienced recombination. However, recombination between the chloroplast genomes of different species would be highly atypical, and corollary supporting observations, like chloroplast heteroplasmy, are lacking. Wider taxon sampling clarified the composition of the Austral group, but the conflicting signal meant analyses (e.g., morphological evolution, biogeographic) conditional on a well-supported phylogeny could not be performed.

The investigation of genetic polymorphisms in the carbonic anhydrase VI gene exon 2 and salivary parameters in type 2 diabetic patients and healthy adults.

Science.gov (United States)

Koç Öztürk, Leyla; Ulucan, Korkut; Akyüz, Serap; Furuncuoğlu, Halit; Bayer, Hikmet; Yarat, Ayşen

2012-05-01

The aim of this study was to investigate carbonic anhydrase (CA) VI Exon 2 single nucleotide polymorphism (SNP) and its possible association with salivary parameters in type 2 diabetic patients compared to healthy adults. Caries status was measured by using the DMFT (number of decayed, missing, and filled teeth) index. Unstimulated whole saliva and blood samples were taken. SNPs of CA gene exon 2 were determined by PCR and DNA sequencing. Salivary CA activity and buffering capacity were determined by the method of Verpoorte and Ericson, respectively. Furthermore, salivary pH was measured with pH paper and salivary flow rate was calculated. Salivary buffering capacity and pH were significantly lower in diabetic patients than those of healthy subjects (P Salivary flow rate, CA activity and DMFT levels did not differ between groups (P > 0.05). Four SNPs were detected; their pubmed database number are rs2274327 (C/T), rs2274328 (A/C), rs2274329 (G/C) and rs2274330. While first three of those were responsible for amino acid changes, the last one was not. The frequencies of SNPs were not significant between groups (P > 0.05). Positive significant correlation was found between CA activity and the frequency of SNPs. There was no correlation between the SNPs frequencies and pH or buffering capacity. SNPs found in this study may be related to salivary CA activity in diabetics.

Peroxisome Proliferator Activated Receptor-α/Hypoxia Inducible Factor-1α Interplay Sustains Carbonic Anhydrase IX and Apoliprotein E Expression in Breast Cancer Stem Cells

Science.gov (United States)

Papi, Alessio; Storci, Gianluca; Guarnieri, Tiziana; De Carolis, Sabrina; Bertoni, Sara; Avenia, Nicola; Sanguinetti, Alessandro; Sidoni, Angelo; Santini, Donatella; Ceccarelli, Claudio; Taffurelli, Mario; Orlandi, Marina; Bonafé, Massimiliano

2013-01-01

Aims Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells “inflammatory addiction” leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells. Methods Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts. Results In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E. Conclusion Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning. PMID:23372804

The complete chloroplast genomes of two Wisteria species, W. floribunda and W. sinensis (Fabaceae).

Science.gov (United States)

Kim, Na-Rae; Kim, Kyunghee; Lee, Sang-Choon; Lee, Jung-Hoon; Cho, Seong-Hyun; Yu, Yeisoo; Kim, Young-Dong; Yang, Tae-Jin

2016-11-01

Wisteria floribunda and Wisteria sinensis are ornamental woody vines in the Fabaceae. The complete chloroplast genome sequences of the two species were generated by de novo assembly using whole genome next generation sequences. The chloroplast genomes of W. floribunda and W. sinensis were 130 960 bp and 130 561 bp long, respectively, and showed inverted repeat (IR)-lacking structures as those reported in IRLC in the Fabaceae. The chloroplast genomes of both species contained same number of protein-coding sequences (77), tRNA genes (30), and rRNA genes (4). The phylogenetic analysis with the reported chloroplast genomes confirmed close taxonomical relationship of W. floribunda and W. sinensis.

Dissecting the chloroplast proteome of chickpea (Cicer arietinum L.) provides new insights into classical and non-classical functions.

Science.gov (United States)

Lande, Nilesh Vikram; Subba, Pratigya; Barua, Pragya; Gayen, Dipak; Keshava Prasad, T S; Chakraborty, Subhra; Chakraborty, Niranjan

2017-08-08

Chloroplast, the energy organelle unique to plant cells, is a dynamic entity which integrates an array of metabolic pathways and serves as first level for energy conversion for the entire ecological hierarchy. Increasing amount of sequence data and evolution of mass spectrometric approaches has opened up new avenues for opportune exploration of the global proteome of this organelle. In our study, we aimed at generation of a comprehensive catalogue of chloroplast proteins in a grain legume, chickpea and provided a reference proteome map. To accurately assign the identified proteins, purity of chloroplast-enriched fraction was stringently monitored by multiple chemical and immunological indexes, besides pigment and enzyme analyses. The proteome analysis led to the identification of 2451 proteins, including 27 isoforms, which include predicted and novel chloroplast constituents. The identified proteins were validated through their sequence analysis. Extensive sequence based localization prediction revealed more than 50% proteins to be chloroplast resident by at least two different algorithms. Chromosomal distribution of identified proteins across nuclear and chloroplast genome unveiled the presence of 55 chloroplast encoded gene. In depth comparison of our dataset with the non-redundant set of chloroplast proteins identified so far across other species revealed novel as well as overlapping candidates. Pulses add large amount of nitrogen to the soil and has very low water footprint and therefore, contributes to fortification of sustainable agriculture. Chickpea is one of the earliest cultivated legumes and serves as an energy and protein source for humans and animals. Chloroplasts are the unique organelles which conduct photosynthesis. Investigation on chloroplast proteome is of particular significance, especially to plant biologists, as it would allow a better understanding of chloroplast function in plants. Generation of a saturated proteome map would not only

Discrete redox signaling pathways regulate photosynthetic light-harvesting and chloroplast gene transcription.

Directory of Open Access Journals (Sweden)

John F Allen

Full Text Available In photosynthesis in chloroplasts, two related regulatory processes balance the actions of photosystems I and II. These processes are short-term, post-translational redistribution of light-harvesting capacity, and long-term adjustment of photosystem stoichiometry initiated by control of chloroplast DNA transcription. Both responses are initiated by changes in the redox state of the electron carrier, plastoquinone, which connects the two photosystems. Chloroplast Sensor Kinase (CSK is a regulator of transcription of chloroplast genes for reaction centres of the two photosystems, and a sensor of plastoquinone redox state. We asked whether CSK is also involved in regulation of absorbed light energy distribution by phosphorylation of light-harvesting complex II (LHC II. Chloroplast thylakoid membranes isolated from a CSK T-DNA insertion mutant and from wild-type Arabidopsis thaliana exhibit similar light- and redox-induced (32P-labelling of LHC II and changes in 77 K chlorophyll fluorescence emission spectra, while room-temperature chlorophyll fluorescence emission transients from Arabidopsis leaves are perturbed by inactivation of CSK. The results indicate indirect, pleiotropic effects of reaction centre gene transcription on regulation of photosynthetic light-harvesting in vivo. A single, direct redox signal is transmitted separately to discrete transcriptional and post-translational branches of an integrated cytoplasmic regulatory system.

RNA transcription in isolated chloroplasts during senescence and rejuvenation of intact cotyledons of CUCURBITA PEPO L. (ZUCCHINI)

International Nuclear Information System (INIS)

Mishev, K.; Ananiev, E.; Denev, L.; Radeva, G.

2006-01-01

RNA transcription was studied in intact chloroplasts isolated from cotyledons of Cucurbita pepoL. (zucchini) during their growth and development including natural senescence and rejuvenation. Rejuvenation of cotyledons was studied after decapitation of the epicotyl above the senescing yellow cotyledons. Maximal incorporation of [32P] UTP into overall chloroplast RNA was measured two days after exposure of seedlings to light (day 6 th after the onset of germination), followed by a gradual decrease reaching minimal values at the age of 25-28 days when cotyledons began to yellow and eventually die. Rejuvenation of cotyledons completely restored chloroplast RNA synthesis and fifteen days after decapitation (at the age of 40 days), the values of chloroplast transcription even exceeded that of the maximal transcriptional activity in young cotyledons. Inhibitory analysis with tagetitoxin (a specific inhibitor of plastid encoded chloroplast RNA polymerase (PEP)) showed that in young and rejuvenated cotyledons about 85% of chloroplast RNA polymerase activity was due to PEP and only 15% corresponded to the nuclear encoded plastid RNA polymerase (NEP). Definite regions of two chloroplast encoded genes were amplified by means of PCR technique using specific DNA primers for Rubisco large subunit gene (rbcL) and the housekeeping gene for chloroplast 16S rRNA as well as chloroplast DNA as a template. The appropriate lengths of the amplified DNA fragments were checked by restriction analysis

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Expression, Folding, Assembly and Functionality

Science.gov (United States)

Chebolu, S.; Daniell, H.

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner. PMID:19401820

Formation and scavenging of superoxide in chloroplasts, with relation to injury by sulfur dioxide

Energy Technology Data Exchange (ETDEWEB)

Asada, K

1980-01-01

Injury of plant leaf cells by sulfur dioxide-exposure is greater in day time than in night. A hypothesis is proposed that the free radical chain oxidation of sulfite is initiated by the superoxide radicals (O/sub 2//sup -/) produced in illuminated chloroplasts, and that the resulting amplified production of O/sub 2//sup -/, the hydroxyl radicals and the bisulfite radicals causes the injury of leaf tissues. In this review, the production of O/sub 2//sup -/ in illuminated chloroplasts and scavenging of O/sub 2//sup -/ by superoxide dismutase and their relation to oxidation of sulfite in chloroplasts are discussed. Superoxide dismutase in chloroplasts plays an important role in protecting leaf cells from injury by sulfur dioxide.

Comparative analyses of chloroplast genome data representing nine green algae in Sphaeropleales (Chlorophyceae, Chlorophyta

Directory of Open Access Journals (Sweden)

Karolina Fučíková

2016-06-01

Full Text Available The chloroplast genomes of green algae are highly variable in their architecture. In this article we summarize gene content across newly obtained and published chloroplast genomes in Chlorophyceae, including new data from nine of species in Sphaeropleales (Chlorophyceae, Chlorophyta. We present genome architecture information, including genome synteny analysis across two groups of species. Also, we provide a phylogenetic tree obtained from analysis of gene order data for species in Chlorophyceae with fully sequenced chloroplast genomes. Further analyses and interpretation of the data can be found in “Chloroplast phylogenomic data from the green algal order Sphaeropleales (Chlorophyceae, Chlorophyta reveal complex patterns of sequence evolution” (Fučíková et al., In review [1].

Insights from the complete chloroplast genome into the evolution of Sesamum indicum L.

Directory of Open Access Journals (Sweden)

Haiyang Zhang

Full Text Available Sesame (Sesamum indicum L. is one of the oldest oilseed crops. In order to investigate the evolutionary characters according to the Sesame Genome Project, apart from sequencing its nuclear genome, we sequenced the complete chloroplast genome of S. indicum cv. Yuzhi 11 (white seeded using Illumina and 454 sequencing. Comparisons of chloroplast genomes between S. indicum and the 18 other higher plants were then analyzed. The chloroplast genome of cv. Yuzhi 11 contains 153,338 bp and a total of 114 unique genes (KC569603. The number of chloroplast genes in sesame is the same as that in Nicotiana tabacum, Vitis vinifera and Platanus occidentalis. The variation in the length of the large single-copy (LSC regions and inverted repeats (IR in sesame compared to 18 other higher plant species was the main contributor to size variation in the cp genome in these species. The 77 functional chloroplast genes, except for ycf1 and ycf2, were highly conserved. The deletion of the cp ycf1 gene sequence in cp genomes may be due either to its transfer to the nuclear genome, as has occurred in sesame, or direct deletion, as has occurred in Panax ginseng and Cucumis sativus. The sesame ycf2 gene is only 5,721 bp in length and has lost about 1,179 bp. Nucleotides 1-585 of ycf2 when queried in BLAST had hits in the sesame draft genome. Five repeats (R10, R12, R13, R14 and R17 were unique to the sesame chloroplast genome. We also found that IR contraction/expansion in the cp genome alters its rate of evolution. Chloroplast genes and repeats display the signature of convergent evolution in sesame and other species. These findings provide a foundation for further investigation of cp genome evolution in Sesamum and other higher plants.

Identification and Characterization of a Chloroplast-Targeted Obg GTPase in Dendrobium officinale.

Science.gov (United States)

Chen, Ji; Deng, Feng; Deng, Mengsheng; Han, Jincheng; Chen, Jianbin; Wang, Li; Yan, Shen; Tong, Kai; Liu, Fan; Tian, Mengliang

2016-12-01

Bacterial homologous chloroplast-targeted Obg GTPases (ObgCs) belong to the plant-typical Obg group, which is involved in diverse physiological processes during chloroplast development. However, the evolutionarily conserved function of ObgC in plants remains elusive and requires further investigation. In this study, we identified DoObgC from an epiphytic plant Dendrobium officinale and demonstrated the characteristics of DoObgC. Sequence analysis indicated that DoObgC is highly conserved with other plant ObgCs, which contain the chloroplast transit peptide (cTP), Obg fold, G domain, and OCT regions. The C terminus of DoObgC lacking the chloroplast-targeting cTP region, DoObgC Δ1-160 , showed strong similarity to ObgE and other bacterial Obgs. Overexpression of DoObgC Δ1-160 in Escherichia coli caused slow cell growth and an increased number of elongated cells. This phenotype was consistent with the phenotype of cells overexpressing ObgE. Furthermore, the expression of recombinant DoObgC Δ1-160 enhanced the cell persistence of E. coli to streptomycin. Results of transient expression assays revealed that DoObgC was localized to chloroplasts. Moreover, we demonstrated that DoObgC could rescue the embryotic lethal phenotype of the Arabidopsis obgc-t mutant, suggesting that DoObgC is a functional homolog to Arabidopsis AtObgC in D. officinale. Gene expression profiles showed that DoObgC was expressed in leaf-specific and light-dependent patterns and that DoObgC responded to wounding treatments. Our previous and present studies reveal that ObgC has an evolutionarily conserved role in ribosome biogenesis to adapt chloroplast development to the environment.

Light-stimulated accumulation of transcripts of nuclear and chloroplast genes for ribulosebisphosphate carboxylase

Energy Technology Data Exchange (ETDEWEB)

Smith, S M; Ellis, R J

1981-01-01

The chloroplast enzyme, ribulosebisphosphate carboxylase, consists of large subunit polypeptides encoded in the chloroplast genome and small subunit polypeptides encoded in the nuclear genome. Cloned DNA complementary to the small subunit mRNA hybridizes to a single RNA species of 900-1000 nucleotides in both total and poly(A)-containing RNA from leaves of Pisum sativum, but does not hybridize to chloroplast RNA. Small subunit cDNA hybridizes to at least three RNA species from nuclei, two of which are of higher molecular weight than the mature mRNA. A cloned large subunit DNA sequence hybridizes to a single species of Pisum chloroplast RNA containing approximately 1700 nucleotides, but does not hybridize to nuclear RNA. The light-stimulation of carboxylase accumulation reflects increases in the amounts of transcripts for both subunits in total leaf RNA. Transcripts of the small subunit gene are more abundant in nuclear RNA from light-grown leaves than in that from dark-grown leaves. These results suggest that the stimulation of carboxylase accumulation by light is mediated at the level of either transcription or RNA turnover in both nucleus and chloroplast.

Carbon isotopes in mollusk shell carbonates

Science.gov (United States)

McConnaughey, Ted A.; Gillikin, David Paul

2008-10-01

Mollusk shells contain many isotopic clues about calcification physiology and environmental conditions at the time of shell formation. In this review, we use both published and unpublished data to discuss carbon isotopes in both bivalve and gastropod shell carbonates. Land snails construct their shells mainly from respired CO2, and shell δ13C reflects the local mix of C3 and C4 plants consumed. Shell δ13C is typically >10‰ heavier than diet, probably because respiratory gas exchange discards CO2, and retains the isotopically heavier HCO3 -. Respired CO2 contributes less to the shells of aquatic mollusks, because CO2/O2 ratios are usually higher in water than in air, leading to more replacement of respired CO2 by environmental CO2. Fluid exchange with the environment also brings additional dissolved inorganic carbon (DIC) into the calcification site. Shell δ13C is typically a few ‰ lower than ambient DIC, and often decreases with age. Shell δ13C retains clues about processes such as ecosystem metabolism and estuarine mixing. Ca2+ ATPase-based models of calcification physiology developed for corals and algae likely apply to mollusks, too, but lower pH and carbonic anhydrase at the calcification site probably suppress kinetic isotope effects. Carbon isotopes in biogenic carbonates are clearly complex, but cautious interpretation can provide a wealth of information, especially after vital effects are better understood.

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.

Chloroplast microsatellites reveal population genetic diversity in red pine, Pinus resinosa Ait

Science.gov (United States)

Craig S. Echt; L.L. DeVerno; M. Anzidei; G.G. Vendramin

1998-01-01

Variation in paternally inherited chloroplast microsatellite (cpSSR) DNA was used to study population genetic structure in red pine (Pinus resinosa Ait.), a species characterized by morphological uniformity, no allozyme variation, and limited RAPD variation. Using nine cpSSR loci, a total of 23 chloroplast haplotypes and 25 cpSSR alleles were were...

Enzymic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria.

Science.gov (United States)

Roberts, M F

1981-08-01

Further studies of the transaminase responsible for the first committed step in alkaloid formation in Conium maculatum have shown the L-alanine: 5-ketooctanal transaminase to occur in both the mitochondria and chloroplast. Experiments suggest that these enzymes are the isoenzymes Transaminase A and B respectively previously isolated by the author. It is suggested that the chloroplast enzyme is normally responsible for alkaloid production.

Chloroplast Signaling Gates Thermotolerance in Arabidopsis

Directory of Open Access Journals (Sweden)

Patrick J. Dickinson

2018-02-01

Full Text Available Temperature is a key environmental variable influencing plant growth and survival. Protection against high temperature stress in eukaryotes is coordinated by heat shock factors (HSFs, transcription factors that activate the expression of protective chaperones such as HEAT SHOCK PROTEIN 70 (HSP70; however, the pathway by which temperature is sensed and integrated with other environmental signals into adaptive responses is not well understood. Plants are exposed to considerable diurnal variation in temperature, and we have found that there is diurnal variation in thermotolerance in Arabidopsis thaliana, with maximal thermotolerance coinciding with higher HSP70 expression during the day. In a forward genetic screen, we identified a key role for the chloroplast in controlling this response, suggesting that light-induced chloroplast signaling plays a key role. Consistent with this, we are able to globally activate binding of HSFA1a to its targets by altering redox status in planta independently of a heat shock.

Photosynthetic assimilation of 14C in isolated chloroplasts in the presence of NO3-, SO4- and NH4+

International Nuclear Information System (INIS)

Tsenova, M.

1977-01-01

Quantitative changes in carbon photosynthesis assimilation occurring as an effect of varying nitrate, sulfate and ammonia ions in the incubation medium were studied in isolated chloroplasts of spinach. Carbon photosynthetic assimilation is enhanced under the influence of rising nitrate anion concentrations to a certain level. The percentage of 14 C concent in the insoluble products is also raised while in glycolic acid it is reduced. The nitrate anion has an effect similar to that of the bicarbonic anion the same processes. Ammonium and sulfate ions have the opposite effect. It can be assumed that the established effect of the ions studied is due to the influence they have on photosynthetic phosphorylation. (author)

Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal beta-class carbonic anhydrase.

Science.gov (United States)

Smith, Kerry S; Ingram-Smith, Cheryl; Ferry, James G

2002-08-01

The roles of an aspartate and an arginine, which are completely conserved in the active sites of beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the beta-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k(cat)/K(m) value depends only on the rate constants for the CO(2) hydration step, whereas k(cat) also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k(cat)/K(m) values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO(2) hydration step. Parallel decreases in k(cat) and k(cat)/K(m) values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k(cat) of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k(cat) and k(cat)/K(m) values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k(cat) of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this

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

Noncoding RNA mediated traffic of foreign mRNA into chloroplasts reveals a novel signaling mechanism in plants.

Directory of Open Access Journals (Sweden)

Gustavo Gómez

Full Text Available Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5'UTR-end mediates the functional import of Green Fluorescent Protein (GFP mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5'UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.

A model for tetrapyrrole synthesis as the primary mechanism for plastid-to-nucleus signaling during chloroplast biogenesis

Directory of Open Access Journals (Sweden)

Matthew J. Terry

2013-02-01

Full Text Available Chloroplast biogenesis involves the co-ordinated expression of the chloroplast and nuclear genomes, requiring information to be sent from the developing chloroplasts to the nucleus. This is achieved through retrograde signaling pathways and can be demonstrated experimentally using the photobleaching herbicide, Norflurazon, which results in chloroplast damage and the reduced expression of many photosynthesis-related, nuclear genes in seedlings. Genetic analysis of this pathway points to a major role for tetrapyrrole synthesis in retrograde signaling, as well as a strong interaction with light-signaling pathways. Currently, the best model to explain the genetic data is that a specific heme pool generated by flux through ferrochelatase-1 functions as a positive signal to promote the expression of genes required for chloroplast development. We propose that this heme-related signal is the primary positive signal during chloroplast biogenesis, and that treatments and mutations affecting chloroplast transcription, RNA editing, translation, or protein import all impact on the synthesis and/or processing of this signal. A positive signal is consistent with the need to provide information on chloroplast status at all times. We further propose that GUN1 normally serves to restrict the production of the heme signal. In addition to a positive signal re-enforcing chloroplast development under normal conditions, aberrant chloroplast development may produce a negative signal due to accumulation of unbound chlorophyll biosynthesis intermediates, such as Mg-porphyrins. Under these conditions a rapid shut-down of tetrapyrrole synthesis is required. We propose that accumulation of these intermediates results in a rapid light-dependent inhibition of nuclear gene expression that is most likely mediated via singlet oxygen generated by photo-excitation of Mg-porphyrins. Thus, the tetrapyrrole pathway may provide both positive and inhibitory signals to control

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

Science.gov (United States)

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

2001-01-01

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

Structure of cells chloroplasts and mitochondria of cotton leaves following gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Arslanova, S V [AN Uzbekskoj SSR, Tashkent. Inst. Ehksperimental' noj Biologii Rastenij

1975-01-01

The article investigates the structural changes in the plastides and mitochondria of cotton leaf cells after irradiation. Cotton seeds that had been moistened for 24 hours were irradiated by a gamma source with a dose of 10 kR (intensity: 19 R/s.). For the study of the plastides and mitochondria of the leaf cells samples were taken in the cotyledonous leaf and flowering phases of the cotton. The cells of the cotton leaf mesophillum in the standard consists of chloroplast with developed interior structures. Study of the ultrastructure of the cells of the mesophilic tissue of the cotyledonous leaf in irradiated cotton plants showed that the chloroplastide membranes are not damaged. A change in the form of the chloroplasts, an accumulation of starch and plastic substances in the chloroplasts, and a reduction in the number of inter-grain bonds were noted. It was discovered that gamma irradiation produces an excessive build-up of starch in the chloroplasts. The mitochondria are often located close to the plastides. The optical density is typical of the matrix of the mitochondria in non-irradiated plants. After cotton seeds that have sprouted are irradiated with a dose of 10 kR in the cotyledonous leaf phase, part of the mitochondria swells. The matrix becomes more transparent, and the number of chrysts decreases. Part of the mitochondria remains intact. The optical density and internal membranes of the mitochondria remain the same as in the control group. The disturbances of the chloroplast and the mitochondria are also observed in the budding and flowering phases (under conditions of a natural day). It was noted that a shortened day facilitated to some extent a normalization of metabolism, and this produced in turn a normal development of the chloroplasts, leaf mitochondria and ATF generation, which reduces the final biological effect of the radiation.

Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Rodermel, Steven

2015-11-16

The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

Science.gov (United States)

Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

2012-01-01

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in C4-related metabolite fluxes and development.

Directory of Open Access Journals (Sweden)

Kalpana eManandhar-Shrestha

2013-03-01

Full Text Available As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second green revolution will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M and bundle sheath (BS chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts (cp. 74% have a known or predicted membrane association. 21 membrane proteins were 2-15 times more abundant in BS cells, while 36 proteins were more abundant in M cp envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of thirteen candidate genes. Cp association was confirmed using GFP labeling. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast

Expression of fungal cutinase and swollenin in tobacco chloroplasts reveals novel enzyme functions and/or substrates.

Directory of Open Access Journals (Sweden)

Dheeraj Verma

Full Text Available In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG and digalactosyldiacylglycerol (DGDG, in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

Science.gov (United States)

Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

2012-11-01

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

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 CO 2 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 CO 2 to HCO 3 - 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 CO 2 . 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 CO 2 exposure. The intracellular pH (pHi) of hemocytes under CO 2 exposure increased significantly (p 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.

Destruction of pigments and lipids in isolated chloroplasts under the effect of visible radiation

International Nuclear Information System (INIS)

Merzlyak, M.N.; Pogosyan, S.I.

1988-01-01

The results of experiments on the effect of light radiation on lipid and pigment destruction in isolated chloroplasts are generalized. Substrates and products of oxidation destruction of lipid and pigments, the role of photosynthetic electron transport in photodestruction, the participation of activated oxygen and free-radical intermediate forms in it are considered. The role of antioxidants, carotenoids and enzymatic systems in protection of chloroplast membranes from destructive light effect is discussed. A general scheme of possible ways of photodestruction in chloroplasts is presented. 53 refs

Enzyme-Triggered Defined Protein Nanoarrays: Efficient Light-Harvesting Systems to Mimic Chloroplasts.

Science.gov (United States)

Zhao, Linlu; Zou, Haoyang; Zhang, Hao; Sun, Hongcheng; Wang, Tingting; Pan, Tiezheng; Li, Xiumei; Bai, Yushi; Qiao, Shanpeng; Luo, Quan; Xu, Jiayun; Hou, Chunxi; Liu, Junqiu

2017-01-24

The elegance and efficiency by which chloroplasts harvest solar energy and conduct energy transfer have been a source of inspiration for chemists to mimic such process. However, precise manipulation to obtain orderly arranged antenna chromophores in constructing artificial chloroplast mimics was a great challenge, especially from the structural similarity and bioaffinity standpoints. Here we reported a design strategy that combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts. Cricoid stable protein one (SP1) was utilized as a building block model. Under enzyme-triggered covalent protein assembly, mutant SP1 with tyrosine (Tyr) residues at the designated sites can couple together to form nanostructures. Through controlling the Tyr sites on the protein surface, we can manipulate the assembly orientation to respectively generate 1D nanotubes and 2D nanosheets. The excellent stability endowed the self-assembled protein architectures with promising applications. We further integrated quantum dots (QDs) possessing optical and electronic properties with the 2D nanosheets to fabricate chloroplast mimics. By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions, we successfully developed an artificial light-harvesting system. The assembled protein nanosheets structurally resembled the natural thylakoids, and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls. Therefore, the coassembled system was meaningful to explore the photosynthetic process in vitro, as it was designed to mimic the natural chloroplast.

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

Science.gov (United States)

Daniell, Henry

2006-10-01

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

A dynamic phase microscopic study of optical characteristics of individual chloroplasts.

Science.gov (United States)

Tychinsky, V P; Kretushev, A V; Vyshenskaya, T V; Tikhonov, A N

2004-10-11

Dynamic phase microscopy (DPM) allows the monitoring of optical path difference (or phase height), h(x,y,t) approximately integraln(x,y,z,t)dz, an integral refractive index projection of the medium, n(x,y,z,t), in optically transparent biological specimens at high spatial and temporal resolutions. In this study, DPM was used for the analysis of fluctuations in the optical characteristics of individual bean chloroplasts in various metabolic states. A "phase image" of an individual chloroplast, which represents a three-dimensional plot of the "phase height", was obtained for the first time, and the frequency spectra of the fluctuations of h(x,y,t) were investigated. The fluctuation patterns, i.e., the intensity and the frequency spectra of phase height fluctuations in bean chloroplasts (Class B) were found to depend on their metabolic state. Under conditions of noncyclic (or pseudocyclic) electron transport, the fluctuations displayed characteristic frequencies in the range of 0.25-0.6 Hz and were space-time-correlated in the chloroplast domains with the cross sizes of approximately 2 microm. The fluctuation intensity decreased in the presence of uncouplers (nigericin and valinomycin, 20 microM). A stronger (in comparison with 20 microM valinomycin) effect of 20 microM nigericin suggests that the light-induced generation of the transmembrane pH difference (DeltapH) makes the main contribution to the increment of space-correlated fluctuations of h(x,y,t). Studies of chloroplasts incubated in media of various osmolarity (50-500 mM sucrose) have shown that structural changes in thylakoids are among other factors responsible for phase height fluctuations.

Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

Science.gov (United States)

Mayfield, Stephen P.

2010-03-16

Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

Fatty acid synthesis by spinach chloroplasts, 2. The path from PGA to fatty acids

Energy Technology Data Exchange (ETDEWEB)

Yamada, Mitsuhiro; Nakamura, Yasunori [Tokyo Univ. (Japan). Coll. of General Education

1975-02-01

By incorporation of /sup 3/H/sub 2/O into the fatty acid chain in the presence of unlabelled precursor, we showed that fatty acids are synthesized from PGA, PEP and pyruvate by intact spinach chloroplasts in the light. /sup 13/C-tracer experiments confirmed that 1-C of pyruvate is decarboxylated and 2-C is incorporated into fatty acids by the chloroplasts. The patterns of fatty acids synthesized from PGA and pyruvate were the same as that from acetate. The highest rate of fatty acid synthesis was reached at the physiological concentration of PGA (3 mM) and pyruvate (1 mM). These results indicate the operation of the following path in the chloroplasts in light: PGA..-->..PEP..-->..pyruvate..-->..acetylCoA..-->..fatty acids. Since citrate and OAA were much less active and malate and glyoxylate were inert as precursors for fatty acid synthesis, PEP or pyruvate carboxylation, citrate lyase reaction and malate synthetase reaction are not involved in the formation of acetylCoA and fatty acids. Since pyruvate was much more effective as a substrate for fatty acid synthesis than lactate, acetaldehyde or acetate, direct decarboxylation path is considered to be the primary path from pyruvate to acetylCoA. The insignificant effect of chloroplast-washing on fatty acid synthesis from PGA and pyruvate indicates that the glycolytic path from PGA to pyruvate is associated with the chloroplasts. Since pyruvate was more effectively incorporated into fatty acids than acetylCoA, it is unlikely that pyruvate decarboxylation to acetylCoA is due to mitochondria contaminating the chloroplast preparation. On the basis of measurements of /sup 3/H/sub 2/O incorporation in the light and dark, the activity of fatty acid synthesis in spincah leaves appears to be shared by the activities in chloroplasts (87%) and other organelles (13%).

Specific and efficient targeting of cyanobacterial bicarbonate transporters to the inner envelope membrane of chloroplasts in Arabidopsis

Directory of Open Access Journals (Sweden)

Susumu eUehara

2016-02-01

Full Text Available Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

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.

Chloroplast-derived vaccine antigens and biopharmaceuticals: protocols for expression, purification, or oral delivery and functional evaluation.

Science.gov (United States)

Singh, N Dolendro; Ding, Yi; Daniell, Henry

2009-01-01

Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile needles, thereby further decreasing their cost. In this chapter, we describe detailed protocols for chloroplast transformation including the construction of chloroplast transformation vectors, delivery of DNA into plant cells using particle bombardment, selection and regeneration of transformants by tissue culture, confirmation of transgene integration into the chloroplast genome and homoplasmy, evaluation of foreign gene expression, purification of foreign protein, or oral delivery via bioencapsulation, functional evaluation using in vitro and in vivo assays, and evaluation of immunity after challenge with pathogens in suitable animal models.

The complete chloroplast genome of a medicinal plant Epimedium koreanum Nakai (Berberidaceae).

Science.gov (United States)

Lee, Jung-Hoon; Kim, Kyunghee; Kim, Na-Rae; Lee, Sang-Choon; Yang, Tae-Jin; Kim, Young-Dong

2016-11-01

Epimedium koreanum is a perennial medicinal plant distributed in Eastern Asia. The complete chloroplast genome sequences of E. koreanum was obtained by de novo assembly using whole genome next-generation sequences. The chloroplast genome of E. koreanum was 157 218 bp in length and separated into four distinct regions such as large single copy region (89 600 bp), small single copy region (17 222 bp) and a pair of inverted repeat regions (25 198 bp). The genome contained a total of 112 genes including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that E. koreanum is most closely related to Berberis bealei, a traditional medicinal plant in the Berberidaceae family.

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay.

Science.gov (United States)

Tengs, T; Bowers, H A; Ziman, A P; Stoecker, D K; Oldach, D W

2001-02-01

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'--3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.

The action spectrum in chloroplast translocation in multilayer leaf cells

Directory of Open Access Journals (Sweden)

Zbigniew Lechowski

2015-01-01

Full Text Available By measurement of light transmittance through a leaf as criterion of chloroplast translocation, the action spectrum of Ajuga reptans was established. In the spectrum obtained, a correction was introduced for leaf autoabsorption calculated on the basis of the Beer-Lambert law. The action spectrum has two maxima: at λ= 375 nm and λ= 481 nm. The range above 502 nm has no significant effect on chloroplast translocation. Comparison with other objects examined demonstrated that in multilayer leaf cells riboflavin seems also to be a photoreceptor active in this process.

Combined analysis of the chloroplast genome and transcriptome of the Antarctic vascular plant Deschampsia antarctica Desv.

Science.gov (United States)

Lee, Jungeun; Kang, Yoonjee; Shin, Seung Chul; Park, Hyun; Lee, Hyoungseok

2014-01-01

Antarctic hairgrass (Deschampsia antarctica Desv.) is the only natural grass species in the maritime Antarctic. It has been researched as an important ecological marker and as an extremophile plant for studies on stress tolerance. Despite its importance, little genomic information is available for D. antarctica. Here, we report the complete chloroplast genome, transcriptome profiles of the coding/noncoding genes, and the posttranscriptional processing by RNA editing in the chloroplast system. The complete chloroplast genome of D. antarctica is 135,362 bp in length with a typical quadripartite structure, including the large (LSC: 79,881 bp) and small (SSC: 12,519 bp) single-copy regions, separated by a pair of identical inverted repeats (IR: 21,481 bp). It contains 114 unique genes, including 81 unique protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Sequence divergence analysis with other plastomes from the BEP clade of the grass family suggests a sister relationship between D. antarctica, Festuca arundinacea and Lolium perenne of the Poeae tribe, based on the whole plastome. In addition, we conducted high-resolution mapping of the chloroplast-derived transcripts. Thus, we created an expression profile for 81 protein-coding genes and identified ndhC, psbJ, rps19, psaJ, and psbA as the most highly expressed chloroplast genes. Small RNA-seq analysis identified 27 small noncoding RNAs of chloroplast origin that were preferentially located near the 5'- or 3'-ends of genes. We also found >30 RNA-editing sites in the D. antarctica chloroplast genome, with a dominance of C-to-U conversions. We assembled and characterized the complete chloroplast genome sequence of D. antarctica and investigated the features of the plastid transcriptome. These data may contribute to a better understanding of the evolution of D. antarctica within the Poaceae family for use in molecular phylogenetic studies and may also help researchers understand the characteristics of the chloroplast

Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

Science.gov (United States)

Piro, Amalia; Serra, Ilia Anna; Spadafora, Antonia; Cardilio, Monica; Bianco, Linda; Perrotta, Gaetano; Santos, Rui; Mazzuca, Silvia

2015-12-01

Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

Directory of Open Access Journals (Sweden)

Jian eWu

2012-11-01

Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

Genetic analysis of a Microseris douglasii (Asteraceae) population polymorphic for an alien chloroplast type

NARCIS (Netherlands)

Roelofs, Dick; Bachmann, Konrad

1997-01-01

Recent evidence suggests chloroplast introgression from Microseris bigelovii into M. douglasii. We have examined 23 plants from a population of M. douglasii polymorphic for M. douglasii and M. bigelovii chloroplast types. All 23 plants were completely homozygous for morphological and RAPD markers,

The different fates of mitochondria and chloroplasts during dark-induced senescence in Arabidopsis leaves.

Science.gov (United States)

Keech, Olivier; Pesquet, Edouard; Ahad, Abdul; Askne, Anna; Nordvall, Dag; Vodnala, Sharvani Munender; Tuominen, Hannele; Hurry, Vaughan; Dizengremel, Pierre; Gardeström, Per

2007-12-01

Senescence is an active process allowing the reallocation of valuable nutrients from the senescing organ towards storage and/or growing tissues. Using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs), we investigated the fate of mitochondria and chloroplasts during dark-induced leaf senescence. Combining in vivo visualization of fates of the two organelles by three-dimensional reconstructions of abaxial parts of leaves with functional measurements of photosynthesis and respiration, we showed that the two experimental systems displayed major differences during 6 d of dark treatment. In whole DPs, organelles were largely retained in both epidermal and mesophyll cells. However, while the photosynthetic capacity was maintained, the capacity of mitochondrial respiration decreased. In contrast, IDLs showed a rapid decline in photosynthetic capacity while maintaining a high capacity for mitochondrial respiration throughout the treatment. In addition, we noticed an unequal degradation of organelles in the different cell types of the senescing leaf. From these data, we suggest that metabolism in leaves of the whole DPs enters a 'stand-by mode' to preserve the photosynthetic machinery for as long as possible. However, in IDLs, mitochondria actively provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. Finally, the heterogeneity of the degradation processes involved during senescence is discussed with regard to the fate of mitochondria and chloroplasts in the different cell types.

Longevity of guard cell chloroplasts in falling leaves: implication for stomatal function and cellular aging.

Science.gov (United States)

Zeiger, E; Schwartz, A

1982-11-12

Guard cell chloroplasts in senescing leaves from 12 species of perennial trees and three species of annual plants survived considerably longer than their mesophyll counterparts. In Ginkgo biloba, stomata from yellow leaves opened during the day and closed at night; guard cell chloroplasts from these leaves showed fluorescence transients associated with electron transport and photophosphorylation. These findings indicate that guard cell chloroplasts are highly conserved throughout the life-span of the leaf and that leaves retain stomatal control during senescence.

Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

Science.gov (United States)

Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

2013-01-01

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

Directory of Open Access Journals (Sweden)

Kwang-Chul Kwon

Full Text Available Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress or paraquat (abiotic stress, GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide, which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

Science.gov (United States)

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit.

Science.gov (United States)

D'Andrea, Lucio; Amenós, Montse; Rodríguez-Concepción, Manuel

2014-01-01

Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiated plastids (chloroplasts and chromoplasts), as well as intermediate stages, in fresh tomato fruits. The method is based on the differential autofluorescence of chlorophylls and carotenoids (lycopene) detected by Confocal Laser Scanning Microscopy.

Arabidopsis CDS blastp result: AK107061 [KOME

Lifescience Database Archive (English)

Full Text Available AK107061 002-121-E01 At3g52720.1 carbonic anhydrase family protein low similarity to storage protein (diosco...rin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 2e-37 ...

Arabidopsis CDS blastp result: AK059525 [KOME

Lifescience Database Archive (English)

Full Text Available AK059525 001-029-D02 At3g52720.1 carbonic anhydrase family protein low similarity to storage protein (diosco...rin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 6e-60 ...

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

Science.gov (United States)

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

Science.gov (United States)

Walker, Joseph F; Zanis, Michael J; Emery, Nancy C

2014-04-01

Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

Science.gov (United States)

Jouhet, Juliette; Gray, John C

2009-10-01

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae.

Science.gov (United States)

Du, Qingwei; Bi, Guiqi; Mao, Yunxiang; Sui, Zhenghong

2016-06-01

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein-coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein-coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya). © 2016 Phycological Society of America.

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 3e-44 ... ...AK241288 J065137F18 At2g28210.1 68415.m03425 carbonic anhydrase family protein simi

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 2e-44 ... ...AK241288 J065137F18 At4g20990.1 68417.m03038 carbonic anhydrase family protein simi

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 5e-38 ... ...AK241288 J065137F18 At4g21000.1 68417.m03039 carbonic anhydrase family protein simi

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 2e-45 ... ...AK241288 J065137F18 At1g08065.1 68414.m00882 carbonic anhydrase family protein simi

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 1e-54 ... ...AK241288 J065137F18 At1g08080.1 68414.m00884 carbonic anhydrase family protein simi

Arabidopsis CDS blastp result: AK241288 [KOME

Lifescience Database Archive (English)

Full Text Available lar to storage protein (dioscorin) [Dioscorea cayenensis] GI:433463; contains Pfam profile PF00194: Eukaryotic-type carbonic anhydrase 6e-45 ... ...AK241288 J065137F18 At5g04180.1 68418.m00406 carbonic anhydrase family protein simi

FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division

Directory of Open Access Journals (Sweden)

Shin-Ya eMiyagishima

2014-09-01

Full Text Available The chloroplast division machinery is a mixture of a stromal FtsZ-based complex descended from a cyanobacterial ancestor of chloroplasts and a cytosolic dynamin-related protein (DRP 5B-based complex derived from the eukaryotic host. Molecular genetic studies have shown that each component of the division machinery is normally essential for normal chloroplast division. However, several exceptions have been found. In the absence of the FtsZ ring, nonphotosynthetic plastids are able to proliferate, likely by elongation and budding. Depletion of DRP5B impairs, but does not stop chloroplast division. Chloroplasts in glaucophytes, which possesses a peptidoglycan (PG layer, divide without DRP5B. Certain parasitic eukaryotes possess nonphotosynthetic plastids of secondary endosymbiotic origin, but neither FtsZ nor DRP5B is encoded in their genomes. Elucidation of the FtsZ- and/or DRP5B-less chloroplast division mechanism will lead to a better understanding of the function and evolution of the chloroplast division machinery and the finding of the as-yet-unknown mechanism that is likely involved in chloroplast division. Recent studies have shown that FtsZ was lost from a variety of prokaryotes, many of which lost PG by regressive evolution. In addition, even some of the FtsZ-bearing bacteria are able to divide when FtsZ and PG are depleted experimentally. In some cases, alternative mechanisms for cell division, such as budding by an increase of the cell surface-to-volume ratio, are proposed. Although PG is believed to have been lost from chloroplasts other than in glaucophytes, there is some indirect evidence for the existence of PG in chloroplasts. Such information is also useful for understanding how nonphotosynthetic plastids are able to divide in FtsZ-depleted cells and the reason for the retention of FtsZ in chloroplast division. Here we summarize information to facilitate analyses of FtsZ- and/or DRP5B-less chloroplast and nonphotosynthetic plastid

Biparental chloroplast inheritance leads to rescue from cytonuclear incompatibility.

Science.gov (United States)

Barnard-Kubow, Karen B; McCoy, Morgan A; Galloway, Laura F

2017-02-01

Although organelle inheritance is predominantly maternal across animals and plants, biparental chloroplast inheritance has arisen multiple times in the angiosperms. Biparental inheritance has the potential to impact the evolutionary dynamics of cytonuclear incompatibility, interactions between nuclear and organelle genomes that are proposed to be among the earliest types of genetic incompatibility to arise in speciation. We examine the interplay between biparental inheritance and cytonuclear incompatibility in Campanulastrum americanum, a plant species exhibiting both traits. We first determine patterns of chloroplast inheritance in genetically similar and divergent crosses, and then associate inheritance with hybrid survival across multiple generations. There is substantial biparental inheritance in C. americanum. The frequency of biparental inheritance is greater in divergent crosses and in the presence of cytonuclear incompatibility. Biparental inheritance helps to mitigate cytonuclear incompatibility, leading to increased fitness of F 1 hybrids and recovery in the F 2 generation. This study demonstrates the potential for biparental chloroplast inheritance to rescue cytonuclear compatibility, reducing cytonuclear incompatibility's contribution to reproductive isolation and potentially slowing speciation. The efficacy of rescue depended upon the strength of incompatibility, with a greater persistence of weak incompatibilities in later generations. These findings suggest that incompatible plastids may lead to selection for biparental inheritance. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta.

Directory of Open Access Journals (Sweden)

Kui Shi

Full Text Available Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs and proteins (DEPs, respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

Separation of Chloroplast Pigments Using Reverse Phase Chromatography.

Science.gov (United States)

Reese, R. Neil

1997-01-01

Presents a protocol that uses reverse phase chromatography for the separation of chloroplast pigments. Provides a simple and relatively safe procedure for use in teaching laboratories. Discusses pigment extraction, chromatography, results, and advantages of the process. (JRH)

Photosynthesis-dependent H2O2 transfer from chloroplasts to nuclei provides a high-light signalling mechanism.

Science.gov (United States)

Exposito-Rodriguez, Marino; Laissue, Pierre Philippe; Yvon-Durocher, Gabriel; Smirnoff, Nicholas; Mullineaux, Philip M

2017-06-29

Chloroplasts communicate information by signalling to nuclei during acclimation to fluctuating light. Several potential operating signals originating from chloroplasts have been proposed, but none have been shown to move to nuclei to modulate gene expression. One proposed signal is hydrogen peroxide (H 2 O 2 ) produced by chloroplasts in a light-dependent manner. Using HyPer2, a genetically encoded fluorescent H 2 O 2 sensor, we show that in photosynthetic Nicotiana benthamiana epidermal cells, exposure to high light increases H 2 O 2 production in chloroplast stroma, cytosol and nuclei. Critically, over-expression of stromal ascorbate peroxidase (H 2 O 2 scavenger) or treatment with DCMU (photosynthesis inhibitor) attenuates nuclear H 2 O 2 accumulation and high light-responsive gene expression. Cytosolic ascorbate peroxidase over-expression has little effect on nuclear H 2 O 2 accumulation and high light-responsive gene expression. This is because the H 2 O 2 derives from a sub-population of chloroplasts closely associated with nuclei. Therefore, direct H 2 O 2 transfer from chloroplasts to nuclei, avoiding the cytosol, enables photosynthetic control over gene expression.Multiple plastid-derived signals have been proposed but not shown to move to the nucleus to promote plant acclimation to fluctuating light. Here the authors use a fluorescent hydrogen peroxide sensor to provide evidence that H 2 O 2 is transferred directly from chloroplasts to nuclei to control nuclear gene expression.

Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide

Directory of Open Access Journals (Sweden)

Leonor ePuerto-Galán

2013-08-01

Full Text Available Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species (ROS, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs, thiol-based peroxidases able to reduce hydrogen- and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide.

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

Science.gov (United States)

Reddick, L Evan; Chotewutmontri, Prakitchai; Crenshaw, Will; Dave, Ashita; Vaughn, Michael; Bruce, Barry D

2008-01-01

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

Science.gov (United States)

Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

2010-06-01

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further

Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of the dinoflagellate Amphidinium carterae

KAUST Repository

Barbrook, Adrian C.; Dorrell, Richard G.; Burrows, Jennifer; Plenderleith, Lindsey J.; Nisbet, R. Ellen R.; Howe, Christopher J.

2012-01-01

-PCR to study transcription and transcript processing in the chloroplasts of Amphidinium carterae, a model peridinin-containing dinoflagellate. These organisms have a highly unusual chloroplast genome, with genes located on multiple small 'minicircle' elements

Contribution of various carbon sources toward isoprene biosynthesis in poplar leaves mediated by altered atmospheric CO2 concentrations.

Directory of Open Access Journals (Sweden)

Amy M Trowbridge

Full Text Available Biogenically released isoprene plays important roles in both tropospheric photochemistry and plant metabolism. We performed a (13CO(2-labeling study using proton-transfer-reaction mass spectrometry (PTR-MS to examine the kinetics of recently assimilated photosynthate into isoprene emitted from poplar (Populus × canescens trees grown and measured at different atmospheric CO(2 concentrations. This is the first study to explicitly consider the effects of altered atmospheric CO(2 concentration on carbon partitioning to isoprene biosynthesis. We studied changes in the proportion of labeled carbon as a function of time in two mass fragments, M41(+, which represents, in part, substrate derived from pyruvate, and M69(+, which represents the whole unlabeled isoprene molecule. We observed a trend of slower (13C incorporation into isoprene carbon derived from pyruvate, consistent with the previously hypothesized origin of chloroplastic pyruvate from cytosolic phosphenolpyruvate (PEP. Trees grown under sub-ambient CO(2 (190 ppmv had rates of isoprene emission and rates of labeling of M41(+ and M69(+ that were nearly twice those observed in trees grown under elevated CO(2 (590 ppmv. However, they also demonstrated the lowest proportion of completely labeled isoprene molecules. These results suggest that under reduced atmospheric CO(2 availability, more carbon from stored/older carbon sources is involved in isoprene biosynthesis, and this carbon most likely enters the isoprene biosynthesis pathway through the pyruvate substrate. We offer direct evidence that extra-chloroplastic rather than chloroplastic carbon sources are mobilized to increase the availability of pyruvate required to up-regulate the isoprene biosynthesis pathway when trees are grown under sub-ambient CO(2.

The complete chloroplast genome sequence of Helwingia himalaica (Helwingiaceae, Aquifoliales) and a chloroplast phylogenomic analysis of the Campanulidae.

Science.gov (United States)

Yao, Xin; Liu, Ying-Ying; Tan, Yun-Hong; Song, Yu; Corlett, Richard T

2016-01-01

Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for Helwingia himalaica , the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large (c. 35,000 species) subclass Campanulidae in order to investigate relationships at the order and family levels. The Helwingia genome consists of 158,362 bp containing a pair of inverted repeat (IR) regions of 25,996 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region which are 87,810 and 18,560 bp, respectively. There are 142 known genes, including 94 protein-coding genes, eight ribosomal RNA genes, and 40 tRNA genes. The topology of the phylogenetic relationships between Apiales, Asterales, and Dipsacales differed between analyses based on complete genome sequences and on 36 shared protein-coding genes, showing that further studies of campanulid phylogeny are needed.

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

Science.gov (United States)

Yao, Xiaohong; Tang, Ping; Li, Zuozhou; Li, Dawei; Liu, Yifei; Huang, Hongwen

2015-01-01

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

Directory of Open Access Journals (Sweden)

Shuai Wang

2016-05-01

Full Text Available Plants have varying abilities to tolerate chilling (low but not freezing temperatures, and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

Carbonic anhydrase inhibitors. Comparison of chlorthalidone, indapamide, trichloromethiazide, and furosemide X-ray crystal structures in adducts with isozyme II, when several water molecules make the difference.

Science.gov (United States)

Temperini, Claudia; Cecchi, Alessandro; Scozzafava, Andrea; Supuran, Claudiu T

2009-02-01

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K(I)s of 65-138 nM), whereas indapamide is a much weaker one (K(I) of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2-3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.

The Complete Chloroplast Genome Sequences of the Medicinal Plant Forsythia suspensa (Oleaceae

Directory of Open Access Journals (Sweden)

Wenbin Wang

2017-10-01

Full Text Available Forsythia suspensa is an important medicinal plant and traditionally applied for the treatment of inflammation, pyrexia, gonorrhea, diabetes, and so on. However, there is limited sequence and genomic information available for F. suspensa. Here, we produced the complete chloroplast genomes of F. suspensa using Illumina sequencing technology. F. suspensa is the first sequenced member within the genus Forsythia (Oleaceae. The gene order and organization of the chloroplast genome of F. suspensa are similar to other Oleaceae chloroplast genomes. The F. suspensa chloroplast genome is 156,404 bp in length, exhibits a conserved quadripartite structure with a large single-copy (LSC; 87,159 bp region, and a small single-copy (SSC; 17,811 bp region interspersed between inverted repeat (IRa/b; 25,717 bp regions. A total of 114 unique genes were annotated, including 80 protein-coding genes, 30 tRNA, and four rRNA. The low GC content (37.8% and codon usage bias for A- or T-ending codons may largely affect gene codon usage. Sequence analysis identified a total of 26 forward repeats, 23 palindrome repeats with lengths >30 bp (identity > 90%, and 54 simple sequence repeats (SSRs with an average rate of 0.35 SSRs/kb. We predicted 52 RNA editing sites in the chloroplast of F. suspensa, all for C-to-U transitions. IR expansion or contraction and the divergent regions were analyzed among several species including the reported F. suspensa in this study. Phylogenetic analysis based on whole-plastome revealed that F. suspensa, as a member of the Oleaceae family, diverged relatively early from Lamiales. This study will contribute to strengthening medicinal resource conservation, molecular phylogenetic, and genetic engineering research investigations of this species.

High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

Directory of Open Access Journals (Sweden)

Wenqin Wang

Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

Formation and Change of Chloroplast-Located Plant Metabolites in Response to Light Conditions

Directory of Open Access Journals (Sweden)

Yiyong Chen

2018-02-01

Full Text Available Photosynthesis is the central energy conversion process for plant metabolism and occurs within mature chloroplasts. Chloroplasts are also the site of various metabolic reactions involving amino acids, lipids, starch, and sulfur, as well as where the production of some hormones takes place. Light is one of the most important environmental factors, acting as an essential energy source for plants, but also as an external signal influencing their growth and development. Plants experience large fluctuations in the intensity and spectral quality of light, and many attempts have been made to improve or modify plant metabolites by treating them with different light qualities (artificial lighting or intensities. In this review, we discuss how changes in light intensity and wavelength affect the formation of chloroplast-located metabolites in plants.

Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

Directory of Open Access Journals (Sweden)

Yoko Kimata-Ariga

Full Text Available Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT, nitrite reductase (NiR and glutamine synthetase (GS, separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE. GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa and multiple sizes (>120 kDa, respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

Chloroplast evolution in the Pinus montezumae complex: a coalescent approach to hybridization.

Science.gov (United States)

Matos, J A; Schaal, B A

2000-08-01

This study addresses the evolutionary history of the chloroplast genomes of two closely related pine species, Pinus hartwegii Lindl. and P. montezumae Lamb (subsect. Ponderosae) using coalescent theory and some of the statistical tools that have been developed from it during the past two decades. Pinus hartwegii and P. montezumae are closely related species in the P. montezumae complex (subsect. Ponderosae) of Mexico and Central America. Pinus hartwegii is a high elevation species, whereas P. montezumae occurs at lower elevations. The two species occur on many of the same mountains throughout Mexico. A total of 350 individuals of P. hartwegii and P. montezumae were collected from Nevado de Colima (Jalisco), Cerro Potosí (Nuevo León), Iztaccihuatl/Popocatepetl (México), and Nevado de Toluca (México). The chloroplast genome of P. hartwegii and P. montezumae was mapped using eight restriction enzymes. Fifty-one different haplotypes were characterized; 38 of 160 restriction sites were polymorphic. Clades of most parsimoniously related chloroplast haplotypes are geographically localized and do not overlap in distribution, and the geographically localized clades of haplotypes include both P. hartwegii and P. montezumae. Some haplotypes in the clades occur in only one of the two species, whereas other haplotypes occur in both species. These data strongly suggest ancient and/or ongoing hybridization between P. hartwegii and P. montezumae and a shared chloroplast genome history within geographic regions of Mexico.

Light affects the chloroplast ultrastructure and post-storage photosynthetic performance of watermelon (Citrullus lanatus) plug seedlings.

Science.gov (United States)

Duan, Qingqing; Jiang, Wu; Ding, Ming; Lin, Ye; Huang, Danfeng

2014-01-01

Watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] plug seedlings were stored at 15°C in the light at a photosynthetic photon flux density of 15 µmol·m(-2)·s(-1) or in darkness for 6 days, to evaluate their chloroplast ultrastructure, and associated photosynthetic characteristics. Storage in the dark caused swelling, disordered granal arrangement, and starch grain disappearance in the chloroplasts. In contrast, the chloroplasts stored in the light were relatively normal. As a result, the light-stored seedlings had a significantly higher chlorophyll content, Fv/Fm, and Pn than did dark-stored seedlings. Regardless of whether the seedlings were stored in light or darkness, the Gs and Ls of the seedlings significantly decreased, while the Ci obviously increased when the Pn decreased after 6 days of storage. This result suggests that the decreased Pn is not solely a stomatal effect, as the effects on the chloroplasts contributed to this photosynthetic inhibition. Six days after transplanting, seedlings that were stored in the light or darkness for 2 or 4 days showed complete recovery of chloroplast ultrastructure, chlorophyll content, Fv/Fm, Gs and Pn. When the storage period increased to 6 days, the dark-stored seedlings had a significantly lower Fv/Fm and Pn than the light-stored and control seedlings 6 days after transplanting, which was mainly ascribed to incomplete recovery of chloroplast ultrastructure. Furthermore, the light-stored seedlings exhibited a significantly higher shoot dry weight during storage and a higher percentage dry weight increase after transplanting than the dark-stored seedlings. These effects were enhanced by prolonged storage (4 to 6 days). This study demonstrated that dim light during storage is beneficial for maintaining chloroplast ultrastructure as well as photosynthetic efficiency in watermelon seedlings, thus contributing to the rapid recovery of post-storage photosynthetic performance, which ensures the transplant quality

Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

Science.gov (United States)

Mitchelson, K R

1996-01-01

The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of the dinoflagellate Amphidinium carterae

KAUST Repository

Barbrook, Adrian C.

2012-05-05

Although transcription and transcript processing in the chloroplasts of plants have been extensively characterised, the RNA metabolism of other chloroplast lineages across the eukaryotes remains poorly understood. In this paper, we use RT-PCR to study transcription and transcript processing in the chloroplasts of Amphidinium carterae, a model peridinin-containing dinoflagellate. These organisms have a highly unusual chloroplast genome, with genes located on multiple small \\'minicircle\\' elements, and a number of idiosyncratic features of RNA metabolism including transcription via a rolling circle mechanism, and 3′ terminal polyuridylylation of transcripts. We demonstrate that transcription occurs in A. carterae via a rolling circle mechanism, as previously shown in the dinoflagellate Heterocapsa, and present evidence for the production of both polycistronic and monocistronic transcripts from A. carterae minicircles, including several regions containing ORFs previously not known to be expressed. We demonstrate the presence of both polyuridylylated and non-polyuridylylated transcripts in A. carterae, and show that polycistronic transcripts can be terminally polyuridylylated. We present a model for RNA metabolism in dinoflagellate chloroplasts where long polycistronic precursors are processed to form mature transcripts. Terminal polyuridylylation may mark transcripts with the correct 3′ end. © 2012 Springer Science+Business Media B.V.

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

Science.gov (United States)

Zeise, Brian; Xu, Danyun; Rappel, Wouter-Jan; Boron, Walter F.; Schroeder, Julian I.

2016-01-01

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

The complete chloroplast genome of Cinnamomum camphora and its comparison with related Lauraceae species

Directory of Open Access Journals (Sweden)

Caihui Chen

2017-09-01

Full Text Available Cinnamomum camphora, a member of the Lauraceae family, is a valuable aromatic and timber tree that is indigenous to the south of China and Japan. All parts of Cinnamomum camphora have secretory cells containing different volatile chemical compounds that are utilized as herbal medicines and essential oils. Here, we reported the complete sequencing of the chloroplast genome of Cinnamomum camphora using illumina technology. The chloroplast genome of Cinnamomum camphora is 152,570 bp in length and characterized by a relatively conserved quadripartite structure containing a large single copy region of 93,705 bp, a small single copy region of 19,093 bp and two inverted repeat (IR regions of 19,886 bp. Overall, the genome contained 123 coding regions, of which 15 were repeated in the IR regions. An analysis of chloroplast sequence divergence revealed that the small single copy region was highly variable among the different genera in the Lauraceae family. A total of 40 repeat structures and 83 simple sequence repeats were detected in both the coding and non-coding regions. A phylogenetic analysis indicated that Calycanthus is most closely related to Lauraceae, both being members of Laurales, which forms a sister group to Magnoliids. The complete sequence of the chloroplast of Cinnamomum camphora will aid in in-depth taxonomical studies of the Lauraceae family in the future. The genetic sequence information will also have valuable applications for chloroplast genetic engineering.

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

Directory of Open Access Journals (Sweden)

Lemieux Claude

2006-02-01

Full Text Available Abstract Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae, in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR featuring an inverted rRNA operon and a small single-copy (SSC region containing 14 genes normally found in the large single-copy (LSC region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of

Clustering of two genes putatively involved in cyanate detoxification evolved recently and independently in multiple fungal lineages

Science.gov (United States)

Fungi that have the enzymes cyanase and carbonic anhydrase show a limited capacity to detoxify cyanate, a fungicide employed by both plants and humans. Here, we describe a novel two-gene cluster that comprises duplicated cyanase and carbonic anhydrase copies, which we name the CCA gene cluster, trac...

Enhanced hydrogen production by coupled system of Halobacterium halobium and chloroplast after entrapment within reverse micelles

Energy Technology Data Exchange (ETDEWEB)

Singh, A.; Dubey, R.S. [Banaras Hindhu University, Varanasi (India). Dept. of Biochemistry; Pandey, K.D. [Banaras Hindhu University, Varanasi (India). Dept. of Botany

1999-08-01

Reverse micelles were used for the enhanced rate of photoproduction of hydrogen using the coupled system of Halobacterium halobium and chloroplasts organelles. Different combinations of organic solvents and surfactants were used for generating reverse micelles. A several fold enhancement in the rate of H{sub 2} production was observed when the coupled system was entrapped within reverse micelles as compared to the aqueous suspension where no detectable H{sub 2} was produced. The coupled system immobilized in reverse micelles formed by sodium lauryl sulfate and carbon tetrachloride yielded maximum rate of H{sub 2} evolution. The optimum temperature for such hydrogen production was 40{sup o}C using light of 520-570 nm wavelength and 100 lux intensity. (author)

Carbonic anhydrase inhibitors. A general approach for the preparation of water-soluble sulfonamides incorporating polyamino-polycarboxylate tails and of their metal complexes possessing long-lasting, topical intraocular pressure-lowering properties.

Science.gov (United States)

Scozzafava, Andrea; Menabuoni, Luca; Mincione, Francesco; Supuran, Claudiu T

2002-03-28

Reaction of polyamino-polycarboxylic acids or their dianhydrides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded mono- and bis-sulfonamides containing polyamino-polycarboxylic acid moieties in their molecule. The acids/anhydrides used in synthesis included IDA, NTA, EDDA, EDTA and EDTA dianhydride, DTPA and DTPA dianhydride, EGTA and EGTA dianhydride, and EDDHA, among others. All the newly prepared derivatives showed strong affinity toward isozymes I, II, and IV of carbonic anhydrase (CA). Metal complexes of the new compounds have also been prepared. Metal ions used in such preparations included di- and trivalent main-group and transition cations, such as Zn(II), Cu(II), Al(III), etc. Some of the new sulfonamides/disulfonamides obtained in this way, as well as their metal complexes, behaved as nanomolar CA inhibitors against isozymes II and IV, being slightly less effective in inhibiting isozyme I. Some of these sulfonamides as well as their metal complexes strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 1-2% water solutions/suspensions. The good water solubility of these sulfonamide CA inhibitors, correlated with the neutral pH of their water solutions used in the ophthalmologic applications and the long duration of action of the IOP-lowering effect, makes them interesting candidates for developing novel types of antiglaucoma drugs devoid of serious topical side effects.

Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.

Science.gov (United States)

Chen, Lih-Jen; Li, Hsou-Min

2017-10-01

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC-TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC-TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC-TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Chloroplast genome resources and molecular markers differentiate rubber dandelion species from weedy relatives.

Science.gov (United States)

Zhang, Yingxiao; Iaffaldano, Brian J; Zhuang, Xiaofeng; Cardina, John; Cornish, Katrina

2017-02-02

Rubber dandelion (Taraxacum kok-saghyz, TK) is being developed as a domestic source of natural rubber to meet increasing global demand. However, the domestication of TK is complicated by its colocation with two weedy dandelion species, Taraxacum brevicorniculatum (TB) and the common dandelion (Taraxacum officinale, TO). TB is often present as a seed contaminant within TK accessions, while TO is a pandemic weed, which may have the potential to hybridize with TK. To discriminate these species at the molecular level, and facilitate gene flow studies between the potential rubber crop, TK, and its weedy relatives, we generated genomic and marker resources for these three dandelion species. Complete chloroplast genome sequences of TK (151,338 bp), TO (151,299 bp), and TB (151,282 bp) were obtained using the Illumina GAII and MiSeq platforms. Chloroplast sequences were analyzed and annotated for all the three species. Phylogenetic analysis within Asteraceae showed that TK has a closer genetic distance to TB than to TO and Taraxacum species were most closely related to lettuce (Lactuca sativa). By sequencing multiple genotypes for each species and testing variants using gel-based methods, four chloroplast Single Nucleotide Polymorphism (SNP) variants were found to be fixed between TK and TO in large populations, and between TB and TO. Additionally, Expressed Sequence Tag (EST) resources developed for TO and TK permitted the identification of five nuclear species-specific SNP markers. The availability of chloroplast genomes of these three dandelion species, as well as chloroplast and nuclear molecular markers, will provide a powerful genetic resource for germplasm differentiation and purification, and the study of potential gene flow among Taraxacum species.

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

Ozone-induced changes in the chloroplast structure of conifer needles, and their use in ozone diagnostics

International Nuclear Information System (INIS)

Kivimaeenpaeae, M.; Sellden, G.; Sutinen, S.

2005-01-01

Ozone induces characteristic symptoms in the chloroplasts of the needles of several coniferous species. Chloroplasts are (1) reduced in size and (2) the stroma is electron dense. Moreover (3) these chloroplast alterations are more pronounced in the outer mesophyll cell layers and in the upper side of the needle compared to the inner layers and lower side. The syndrome, including the three symptoms (1)-(3), is found in the green needles of Scots pine and Norway spruce not only in the experimental fumigations, but also in mature trees in the field, and has potential for diagnosis of ozone stress. For sound ozone diagnostics all three symptoms must be present in the samples studied. The symptoms in relation to needle anatomy and physiology is discussed, and recommendations for sampling and analysis are given. - Ozone-induced alterations in chloroplast structure of conifer needles are reviewed, and recommendations for field monitoring given

Complete chloroplast genome of Trachelium caeruleum: extensiverearrangements are associated with repeats and tRNAs

Energy Technology Data Exchange (ETDEWEB)

Haberle, Rosemarie C.; Fourcade, Matthew L.; Boore, Jeffrey L.; Jansen, Robert K.

2006-01-09

Chloroplast genome structure, gene order and content arehighly conserved in land plants. We sequenced the complete chloroplastgenome sequence of Trachelium caeruleum (Campanulaceae) a member of anangiosperm family known for highly rearranged chloroplast genomes. Thetotal genome size is 162,321 bp with an IR of 27,273 bp, LSC of 100,113bp and SSC of 7,661 bp. The genome encodes 115 unique genes, with 19duplicated in the IR, a tRNA (trnI-CAU) duplicated once in the LSC and aprotein coding gene (psbJ) duplicated twice, for a total of 137 genes.Four genes (ycf15, rpl23, infA and accD) are truncated and likelynonfunctional; three others (clpP, ycf1 and ycf2) are so highly divergedthat they may now be pseudogenes. The most conspicuous feature of theTrachelium genome is the presence of eighteen internally unrearrangedblocks of genes that have been inverted or relocated within the genome,relative to the typical gene order of most angiosperm chloroplastgenomes. Recombination between repeats or tRNAs has been suggested as twomeans of chloroplast genome rearrangements. We compared the relativenumber of repeats in Trachelium to eight other angiosperm chloroplastgenomes, and evaluated the location of repeats and tRNAs in relation torearrangements. Trachelium has the highest number and largest repeats,which are concentrated near inversion endpoints or other rearrangements.tRNAs occur at many but not all inversion endpoints. There is likely nosingle mechanism responsible for the remarkable number of alterations inthis genome, but both repeats and tRNAs are clearly associated with theserearrangements. Land plant chloroplast genomes are highly conserved instructure, gene order and content. The chloroplast genomes of ferns, thegymnosperm Ginkgo, and most angiosperms are nearly collinear, reflectingthe gene order in lineages that diverged from lycopsids and the ancestralchloroplast gene order over 350 million years ago (Raubeson and Jansen,1992). Although earlier mapping studies

GENETIC POLYMORPHISM IN GYMNODINIUM GALATHEANUM CHLOROPLAST DNA SEQUENCES AND DEVELOPMENT OF A MOLECULAR DETECTION ASSAY. (R827084)

Science.gov (United States)

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtainedfrom several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses andcomparison of sequences indicate that the chloroplast sequences show a higher degree of se...

Abscisic acid represses the transcription of chloroplast genes

Czech Academy of Sciences Publication Activity Database

Yamburenko, M.V.; Zubo, Y.O.; Vaňková, Radomíra; Kusnetsov, V.; Kulaeva, O.N.; Borner, T.

2013-01-01

Roč. 64, č. 14 (2013), s. 4491-4502 ISSN 0022-0957 R&D Projects: GA ČR GA522/09/2058 Institutional research plan: CEZ:AV0Z50380511 Keywords : Abscisic acid (ABA) * chloroplast * cytokinin Subject RIV: ED - Physiology Impact factor: 5.794, year: 2013

The complete chloroplast genome sequence of Helwingia himalaica (Helwingiaceae, Aquifoliales and a chloroplast phylogenomic analysis of the Campanulidae

Directory of Open Access Journals (Sweden)

Xin Yao

2016-11-01

Full Text Available Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for Helwingia himalaica, the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large (c. 35,000 species subclass Campanulidae in order to investigate relationships at the order and family levels. The Helwingia genome consists of 158,362 bp containing a pair of inverted repeat (IR regions of 25,996 bp separated by a large single-copy (LSC region and a small single-copy (SSC region which are 87,810 and 18,560 bp, respectively. There are 142 known genes, including 94 protein-coding genes, eight ribosomal RNA genes, and 40 tRNA genes. The topology of the phylogenetic relationships between Apiales, Asterales, and Dipsacales differed between analyses based on complete genome sequences and on 36 shared protein-coding genes, showing that further studies of campanulid phylogeny are needed.

Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

Directory of Open Access Journals (Sweden)

Hongyu Chen

2018-02-01

Full Text Available In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem, cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

OpenAIRE

Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

1990-01-01

During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to ...

A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants

Directory of Open Access Journals (Sweden)

Pląder Wojciech

2011-09-01

Full Text Available Abstract Plastids are small organelles equipped with their own genomes (plastomes. Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile.

Combined effects of simulated acid rain and lanthanum chloride on chloroplast structure and functional elements in rice.

Science.gov (United States)

Hu, Huiqing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-05-01

Acid rain and rare earth element (REE) pollution exist simultaneously in many agricultural regions. However, how REE pollution and acid rain affect plant growth in combination remains largely unknown. In this study, the combined effects of simulated acid rain and lanthanum chloride (LaCl3) on chloroplast morphology, chloroplast ultrastructure, functional element contents, chlorophyll content, and the net photosynthetic rate (P n) in rice (Oryza sativa) were investigated by simulating acid rain and rare earth pollution. Under the combined treatment of simulated acid rain at pH 4.5 and 0.08 mM LaCl3, the chloroplast membrane was smooth, proteins on this membrane were uniform, chloroplast structure was integrated, and the thylakoids were orderly arranged, and simulated acid rain and LaCl3 exhibited a mild antagonistic effect; the Mg, Ca, Mn contents, the chlorophyll content, and the P n increased under this combined treatment, with a synergistic effect of simulated acid rain and LaCl3. Under other combined treatments of simulated acid rain and LaCl3, the chloroplast membrane surface was uneven, a clear "hole" was observed on the surface of chloroplasts, and the thylakoids were dissolved and loose; and the P n and contents of functional elements (P, Mg, K, Ca, Mn, Fe, Ni, Cu, Zn and Mo) and chlorophyll decreased. Under these combined treatments, simulated acid rain and LaCl3 exhibited a synergistic effect. Based on the above results, a model of the combined effects of simulated acid rain and LaCl3 on plant photosynthesis was established in order to reveal the combined effects on plant photosynthesis, especially on the photosynthetic organelle-chloroplast. Our results would provide some references for further understanding the mechanism of the combined effects of simulated acid rain and LaCl3 on plant photosynthesis.

Functional characterization of recombinant chloroplast signal recognition particle

NARCIS (Netherlands)

Groves, M R; Mant, A; Kuhn, A; Koch, J; Dübel, S; Robinson, C; Sinning, I

2001-01-01

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified

Study of the interaction of cytochrome c and ferredoxine with the double membrane of chloroplast

International Nuclear Information System (INIS)

Neuburger, M.; Joyard, J.; Douce, R.

1975-01-01

The adsorption of two 59 Fe-labelled proteins on the chloroplast envelope was studied. The former molecule used was ferredoxine extracted from spinach leaves, the latter was cytochrome c, extracted from yeast (Saccharomyces cerevisiae D 261). The chloroplast envelope is thought to be involved in the transport of some proteins such as ferredoxine synthetized in the cytoplasm [fr

The complete chloroplast genome sequence of the medicinal plant Andrographis paniculata.

Science.gov (United States)

Ding, Ping; Shao, Yanhua; Li, Qian; Gao, Junli; Zhang, Runjing; Lai, Xiaoping; Wang, Deqin; Zhang, Huiye

2016-07-01

The complete chloroplast genome of Andrographis paniculata, an important medicinal plant with great economic value, has been studied in this article. The genome size is 150,249 bp in length, with 38.3% GC content. A pair of inverted repeats (IRs, 25,300 bp) are separated by a large single copy region (LSC, 82,459 bp) and a small single-copy region (SSC, 17,190 bp). The chloroplast genome contains 114 unique genes, 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In these genes, 15 genes contained 1 intron and 3 genes comprised of 2 introns.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

Science.gov (United States)

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

Carbon dioxide capture using resin-wafer electrodeionization

Science.gov (United States)

Lin, YuPo J.; Snyder, Seth W.; Trachtenberg, Michael S.; Cowan, Robert M.; Datta, Saurav

2015-09-08

The present invention provides a resin-wafer electrodeionization (RW-EDI) apparatus including cathode and anode electrodes separated by a plurality of porous solid ion exchange resin wafers, which when in use are filled with an aqueous fluid. The apparatus includes one or more wafers comprising a basic ion exchange medium, and preferably includes one or more wafers comprising an acidic ion exchange medium. The wafers are separated from one another by ion exchange membranes. The fluid within the acidic and/or basic ion exchange wafers preferably includes, or is in contact with, a carbonic anhydrase (CA) enzyme to facilitate conversion of bicarbonate ion to carbon dioxide within the acidic medium. A pH suitable for exchange of CO.sub.2 is electrochemically maintained within the basic and acidic ion exchange wafers by applying an electric potential across the cathode and anode.

Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

Directory of Open Access Journals (Sweden)

Catalina Perello

Full Text Available Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS and reductoisomerase (DXR, can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

Science.gov (United States)

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

Proton gradients and proton-dependent transport processes in the chloroplast

Directory of Open Access Journals (Sweden)

Ricarda eHöhner

2016-02-01

Full Text Available Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7 and the stroma (pH 8 is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+ or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function.

Chloroplast Movement May Impact Plant Phenotyping and Photochemistry Results

Science.gov (United States)

Malas, J.; Pleban, J. R.; Wang, D. R.; Riley, C.; Mackay, D. S.

2017-12-01

Investigating phenotypic responses of crop species across environmental conditions is vital to improving agricultural productivity. Crop production is closely linked with photosynthetic activity, which can be evaluated using parameters such as relative chlorophyll, SPAD, and variable chlorophyll fluorescence. Recently, a handheld device known as the MultispeQ emerged on the market as an open-source instrument that aims to provide high-output, high-quality field data at a low cost to the plant research community. MultispeQ takes measurements of both environmental conditions (light intensity, temperature, humidity, etc.) and photosynthetic parameters (relative chlorophyll, SPAD, photosystem II quantum efficiency (FII), and non-photochemical quenching (NPQ)). Data are automatically backed up and shared on the PhotosynQ network, which serves as a collaborative platform for researchers and professionals. Here, we used the instrument to quantify photosynthetic time-courses of two Brassica rapa genotypes in response to two contrasting nutrient management strategies (Control; High Nitrogen). Previous research found that chloroplast movement is one strategy plants use to optimize photosynthesis across varying light conditions. We were able to detect chloroplast movement throughout the day using the MultispeQ device. Our results support the idea that chloroplast movement serves both as an intrinsic feature of the circadian clock and as a light avoidance strategy. Under low light conditions (PAR 0-300) more light at the near-infrared and red regions was absorbed than under higher light conditions (PAR 500-800). In one genotype by treatment combination, absorbance at 730nm was around 60% at low light, versus only 30% at high light conditions. In light of our results that relative chlorophyll may change throughout a day, we suggest that it is important to take note of these effects when collecting photosynthesis efficiency data in order to avoid bias in measurements. We also

Characterization of polymorphic SSRs among Prunus chloroplast genomes

Science.gov (United States)