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.

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

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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.

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 Institutional support: RVO:61388963 Keywords : human carbonic anhydrase * isoquinoline * quinoline * X-ray * molecular docking Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2015

Optimization of nutritional constituents for carbonic anhydrase ...

African Journals Online (AJOL)

Jane

2011-08-08

Aug 8, 2011 ... for the optimization of the culture media are to select the optimum .... Effect of different temperature on product of carbonic anhydrase. production, B. ... account that the enzyme is easy to inactivate under high temperature ...

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

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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.

Carbonic anhydrase inhibition increases retinal oxygen tension and dilates retinal vessels

DEFF Research Database (Denmark)

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

2005-01-01

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

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

DEFF Research Database (Denmark)

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

In this study the effect of carbonic anhydrase addition on the absorption of CO2 was investigated in a wetted wall column apparatus. Four different solvents: MEA (a primary amine), AMP (a sterically hindered primary amine), MDEA (a tertiary amine) and K2CO3 a carbonate salt solution were tested...

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.

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

DEFF Research Database (Denmark)

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

1999-01-01

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

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

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.

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....

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.

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.

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

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

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.

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

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

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

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...

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.

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.

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

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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

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.

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

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…

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.

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.

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.

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

Directory of Open Access Journals (Sweden)

Marina V. Vodka

2013-04-01

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

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.

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

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.

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.

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.

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.

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.

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.

Carbonic anhydrase activity of integral-functional complexes of thylakoid membranes of spinach chloroplasts

Directory of Open Access Journals (Sweden)

A. V. Semenihin

2015-06-01

Full Text Available Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1 within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1. Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

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.)

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.

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.

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...

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

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.

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.

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.

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.

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.

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.

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

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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

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.

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 ...

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.

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.

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.

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

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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.

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

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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.

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

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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

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

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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.

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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.

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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

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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.

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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.

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

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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.

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

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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.

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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.

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

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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

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

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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

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

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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.

Deficiency of Carbonic Anhydrase II Results in a Urinary Concentrating Defect

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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.

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

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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.

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

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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.

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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.

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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

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

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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

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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.

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

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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.

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

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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

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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

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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.

Binding properties of a streptavidin layer formed on a biotinylated Langmuir–Schaefer film of unfolded protein

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Furuno, Taiji, E-mail: t_furuno@a8.keio.jp

2016-04-01

A Langmuir monolayer of carbonic anhydrase (CA) unfolded at an air/water interface was transferred onto the hydrophobic surface of a silicon wafer by means of the Langmuir–Schaefer technique. The transferred CA film was biotinylated and was incubated in a streptavidin (SAv) solution to obtain a densely packed SAv layer by biotin–SAv linkage. Biotinylated proteins including ferritin, catalase, alcohol dehydrogenase, and carbonic anhydrase were incubated with the SAv layer and binding of these proteins was examined by atomic force microscopy. High-density binding of the biotinylated proteins was observed, whereas the amount of adsorbed non-biotinylated proteins was low or negligible. The SAv layer on the Langmuir–Schaefer film of unfolded protein could become a basic architecture for protein immobilization studies. - Highlights: • Langmuir–Schaefer film of carbonic anhydrase (LSF-CA) was biotinylated. • A densely packed streptavidin (SAv) layer was formed on the biotinylated LSF-CA. • Biotinylated proteins were bound to the SAv layer at high density. • Nonspecific adsorption of intact proteins to the SAv layer was weak. • Atomic force microscopy showed the binding of proteins at molecular resolution.

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

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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.

Cancer Drug Development of Carbonic Anhydrase Inhibitors beyond the Active Site

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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.

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

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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.

Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

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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.

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

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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.

Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

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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

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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.

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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.

Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation.

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Jillette, Nathaniel; Cammack, Lauren; Lowenstein, Margaret; Henry, Raymond P

2011-02-01

The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins. Published by Elsevier Inc.

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

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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

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

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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.

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

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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.

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

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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.

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

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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.

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

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Ian M Sandeman

2012-03-01

, p<0.05 indicating a dependence on carbonate. At a pH of 6.5 the skeleton lost weight at a rate of 1.8 mg.h-1.cm-2. The relationship between net calcification and pH (n=2 indicates that wt gain turns to loss at pH 7.4. These experiments confirm that calcification is a two-step process, involving secretion of a layer of organic matrix incorporating carbonic anhydrase to produce an active calcifying surface which uses carbon dioxide rather than carbonate. It is also unlikely that the calcifying surface is in direct contact with seawater. Inorganic deposition or dissolution of the skeleton in exposed dead areas of coral is a different phenomenon and is carbonate related. The wide range in results from this and other studies of calcification rate and carbon dioxide may be explainable in terms of the ratio of “live” to “dead” areas of coral.

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.

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas.

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Song, Xiaorui; Wang, Xiaotong; Li, Li; Zhang, Guofan

2014-07-01

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.

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.

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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.

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

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Stefan Burén

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

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

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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.

Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells.

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Parks, Scott K; Cormerais, Yann; Durivault, Jerome; Pouyssegur, Jacques

2017-02-07

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pHi regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pHi regulator Na+/H+ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH4Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pHi. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.

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

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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.

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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.

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

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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

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

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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.)

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

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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.

The Evolutionary History of Daphniid α-Carbonic Anhydrase within Animalia

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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)

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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.

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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.

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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.

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

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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

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

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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.

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

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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.

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.

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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).

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

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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.

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

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Nurcan Berber

2013-01-01

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

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

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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.

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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

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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.

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

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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.

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

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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.

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

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Pawel Sadlecki

2014-01-01

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

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

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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.

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

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Dalila Jacqueline Escudero-Almanza

2012-03-01

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

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

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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.

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

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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.

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

Identification of altered plasma proteins by proteomic study in valvular heart diseases and the potential clinical significance.

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Ge Gao

Full Text Available BACKGROUND: Little is known about genetic basis and proteomics in valvular heart disease (VHD including rheumatic (RVD and degenerative (DVD valvular disease. The present proteomic study examined the hypothesis that certain proteins may be associated with the pathological changes in the plasma of VHD patients. METHODS AND RESULTS: Differential protein analysis in the plasma identified 18 differentially expressed protein spots and 14 corresponding proteins or polypeptides by two-dimensional electrophoresis and mass spectrometry in 120 subjects. Two up-regulated (complement C4A and carbonic anhydrase 1 and three down-regulated proteins (serotransferrin, alpha-1-antichymotrypsin, and vitronectin were validated by ELISA in enlarging samples. The plasma levels (n = 40 for each of complement C4A in RVD (715.8±35.6 vs. 594.7±28.2 ng/ml, P = 0.009 and carbonic anhydrase 1 (237.70±15.7 vs. 184.7±10.8 U/L, P = 0.007 in DVD patients were significantly higher and that of serotransferrin (2.36±0.20 vs. 2.93±0.16 mg/ml, P = 0.025 and alpha-1-antichymotrypsin (370.0±13.7 vs. 413.0±11.6 µg/ml, P = 0.019 in RVD patients were significantly lower than those in controls. The plasma vitronectin level in both RVD (281.3±11.0 vs. 323.2±10.0 µg/ml, P = 0.006 and DVD (283.6±11.4 vs. 323.2±10.0 µg/ml, P = 0.011 was significantly lower than those in normal controls. CONCLUSIONS: We have for the first time identified alterations of 14 differential proteins or polypeptides in the plasma of patients with various VHD. The elevation of plasma complement C4A in RVD and carbonic anhydrase 1 in DVD and the decrease of serotransferrin and alpha-1-antichymotrypsin in RVD patients may be useful biomarkers for these valvular diseases. The decreased plasma level of vitronectin - a protein related to the formation of valvular structure - in both RVD and DVD patients might indicate the possible genetic deficiency in these patients.

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....

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

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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.

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

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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.

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

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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

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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

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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

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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.

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

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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.

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

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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.

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

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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.

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

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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

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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.

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

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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.

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

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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.

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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.

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

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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

CO2 sequestration using principles of shell formation

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Lee, Seung-Woo; Jang, Young-Nam [CO2 Sequestration Research Department, Korea Institute of Geoscience and Mineral Resources (Korea, Republic of); Lee, Si-Hyun; Lim, Kyoung-Soo; Jeong, Soon-Kwan [Energy Conservation Research Department of Clean Energy System Research Center, Korea Institute of Energy Research (Korea, Republic of)

2011-06-15

The biomimetic sequestration of carbon dioxide to reduce the CO2 emitted into the atmosphere is introduced in this paper. Bivalve shells are used as a good model of CO2 sequestration in this paper, because the shell is derived from the calcium ions and CO2 in seawater. Carbonic anhydrase, hemocyte from diseased shell (HDS) and extrapallial fluid (EFP) are involved in shell formation. This paper compares the soluble protein extracted from Crassostrea gigas with bovine carbonic anhydrase II in terms of their ability to promote CO2 hydration and the production of calcium precipitates. The result demonstrates that HDS has more functional groups to bind calcium ions in aqueous systems, and a different process of calcium precipitation, than does bovine carbonic anhydrase II. To understand molecular weight and secondary protein structure, mass-spectroscopic analysis (MALDI-TOF) and circular dichroism (CD) analysis were used. With regard to EPF, EPF related to shell formation is composed of several fractions and plays a role in sequestration of CO2.

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

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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.

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

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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

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.

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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.

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

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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.

Tuber storage proteins.

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Shewry, Peter R

2003-06-01

A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

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

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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.

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

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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.

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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.

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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.

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

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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

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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

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

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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.

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

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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

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.

Carbon allocation and element composition in four Chlamydomonas mutants defective in genes related to the CO2 concentrating mechanism.

Science.gov (United States)

Memmola, Francesco; Mukherjee, Bratati; Moroney, James V; Giordano, Mario

2014-09-01

Four mutants of Chlamydomonas reinhardtii with defects in different components of the CO2 concentrating mechanism (CCM) or in Rubisco activase were grown autotrophically at high pCO2 and then transferred to low pCO2, in order to study the role of different components of the CCM on carbon allocation and elemental composition. To study carbon allocation, we measured the relative size of the main organic pools by Fourier Transform Infrared spectroscopy. Total reflection X-ray fluorescence was used to analyze the elemental composition of algal cells. Our data show that although the organic pools increased their size at high CO2 in all strains, their stoichiometry was highly homeostatic, i.e., the ratios between carbohydrates and proteins, lipid and proteins, and carbohydrates and lipids, did not change significantly. The only exception was the wild-type 137c, in which proteins decreased relative to carbohydrates and lipids, when the cells were transferred to low CO2. It is noticeable that the two wild types used in this study responded differently to the transition from high to low CO2. Malfunctions of the CCM influenced the concentration of several elements, somewhat altering cell elemental stoichiometry: especially the C/P and N/P ratios changed appreciably in almost all strains as a function of the growth CO2 concentration, except in 137c and the Rubisco activase mutant rca1. In strain cia3, defective in the lumenal carbonic anhydrase (CA), the cell quotas of P, S, Ca, Mn, Fe, and Zn were about 5-fold higher at low CO2 than at high CO2. A Principle Components Analysis showed that, mostly because of its elemental composition, cia3 behaved in a substantially different way from all other strains, at low CO2. The lumenal CA thus plays a crucial role, not only for the correct functioning of the CCM, but also for element utilization. Not surprisingly, growth at high CO2 attenuated differences among strains.

In vitro hydroxyapatite adsorbed salivary proteins

International Nuclear Information System (INIS)

Vitorino, Rui; Lobo, Maria Joao C.; Duarte, Jose; Ferrer-Correia, Antonio J.; Tomer, Kenneth B.; Dubin, Joshua R.; Domingues, Pedro M.; Amado, Francisco M.L.

2004-01-01

In spite of the present knowledge about saliva components and their respective functions, the mechanism(s) of pellicle and dental plaque formation have hitherto remained obscure. This has prompted recent efforts on in vitro studies using hydroxyapatite (HA) as an enamel model. In the present study salivary proteins adsorbed to HA were extracted with TFA and EDTA and resolved by 2D electrophoresis over a pH range between 3 and 10, digested, and then analysed by MALDI-TOF/TOF mass spectrometry and tandem mass spectrometry. Nineteen different proteins were identified using automated MS and MS/MS data acquisition. Among them, cystatins, amylase, carbonic anhydrase, and calgranulin B, were identified

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.

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

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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

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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.

Participatory role of zinc in structural and functional characterization of bioremediase: a unique thermostable microbial silica leaching protein.

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Chowdhury, Trinath; Sarkar, Manas; Chaudhuri, Biswadeep; Chattopadhyay, Brajadulal; Halder, Umesh Chandra

2015-07-01

A unique protein, bioremediase (UniProt Knowledgebase Accession No.: P86277), isolated from a hot spring bacterium BKH1 (GenBank Accession No.: FJ177512), has shown to exhibit silica leaching activity when incorporated to prepare bio-concrete material. Matrix-assisted laser desorption ionization mass spectrometry analysis suggests that bioremediase is 78% homologous to bovine carbonic anhydrase II though it does not exhibit carbonic anhydrase-like activity. Bioinformatics study is performed for understanding the various physical and chemical parameters of the protein which predicts the involvement of zinc encircled by three histidine residues (His94, His96 and His119) at the active site of the protein. Isothermal titration calorimetric-based thermodynamic study on diethyl pyrocarbonate-modified protein recognizes the presence of Zn(2+) in the enzyme moiety. Exothermic to endothermic transition as observed during titration of the protein with Zn(2+) discloses that there are at least two binding sites for zinc within the protein moiety. Addition of Zn(2+) regains the activity of EDTA chelated bioremediase confirming the presence of extra binding site of Zn(2+) in the protein moiety. Revival of folding pattern of completely unfolded urea-treated protein by Zn(2+) explains the participatory role of zinc in structural stability of the protein. Restoration of the λ max in intrinsic fluorescence emission study of the urea-treated protein by Zn(2+) similarly confirms the involvement of Zn in the refolding of the protein. The utility of bioremediase for silica nanoparticles preparation is observed by field emission scanning electron microscopy.

Photoaggregation of crystallins (main proteins of eye lens) under the effect of XeCl laser radiation

Science.gov (United States)

Soustov, Lev V.; Chelnokov, Evgeny V.; Bityurin, Nikita M.; Kiselev, A. L.; Nemov, V. V.; Sergeev, Yu. V.; Ostrovsky, Michail A.

2004-07-01

UV light is one of primary factors associated with cataract formation in the eye lens. α-, β-, γ-Crystallins maintain lens transparency, and damage to these proteins plays a major role in cataract formation. The effect of XeCl laser radiation (308 nm) on βL-crystallin solution is studied. The strong dependence of protein aggregation kinetics on both laser fluence (w) and repetition rate (F) is investigated. The kinetics features are similar to those of carbonic anhydrase photoaggregation studied previously.

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.

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.

Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation

Science.gov (United States)

Barshishat-Kupper, Michal; McCart, Elizabeth A.; Freedy, James G.; Tipton, Ashlee J.; Nagy, Vitaly; Kim, Sung-Yop; Landauer, Michael R.; Mueller, Gregory P.; Day, Regina M.

2015-01-01

Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure. PMID:28248270

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).

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.

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.

Protein (Viridiplantae): 94306 [PGDBj - Ortholog DB

Lifescience Database Archive (English)

Full Text Available 47368:3137 ... 147385:3137 ... 15367:3137 ... 15368:3137 ... PREDICTED: bifunctional monodehydroascorbate reductase and carbonic anhydrase nect...arin-3-like Brachypodium distachyon MATRVGNAVVFALLLCARFL

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

Bilateral Intravitreal Dexamethasone Implant for Retinitis Pigmentosa-Related Macular Edema

Directory of Open Access Journals (Sweden)

Ali Osman Saatci

2013-03-01

Full Text Available Purpose: To report the efficacy of intravitreal dexamethasone implant in a patient with retinitis pigmentosa and bilateral cystoid macular edema unresponsive to topical carbonic anhydrase inhibitors. Case Report: A 36-year-old man with bilateral cystoid macular edema associated with retinitis pigmentosa that was unresponsive to topical carbonic anhydrase inhibitors underwent bilateral 0.7-mg intravitreal dexamethasone implants two weeks apart. Spectral domain optical coherence tomography revealed resolution of macular edema one week following each injection in both eyes and his visual acuity improved. However, macular edema recurred two months later in OS and three months later in OD. Second implant was considered for both eyes. No implant-related complication was experienced during the follow-up of seven months. Conclusion: Inflammatory process seems to play a role in retinitis pigmentosa. Intravitreal dexamethasone implant may offer retina specialists a therapeutic option especially in cases unresponsive to other treatment regimens in eyes with retinitis pigmentosa-related macular edema.

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

Cloning of genes and enzymatic characterizations of novel dioscorin isoforms from Dioscorea japonica.

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Xue, You-Lin; Miyakawa, Takuya; Sawano, Yoriko; Tanokura, Masaru

2012-02-01

Dioscorin, the major tuber storage protein of yam, has been shown to possess carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. In the present study, dioscorin from Dioscorea japonica was confirmed as a glycoprotein using the enhanced concanavalin A-peroxidase staining method, and the protein was shown to have both N- and O-glycans. Following the gene cloning, four full-length isoforms of dioscorin were expressed in Escherichia coli and purified by affinity purification and anion-exchange chromatography for structural and biochemical experiments. It was clearly observed that the recombinant dioscorins had carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. However, the dehydroascorbate reductase and monodehydroascorbate reductase activities were markedly decreased in recombinant dioscorins compared with native dioscorin. The decreased activities were closely related to the loss of the glycosylation from the protein. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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.

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.

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

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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.

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

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Mayank Aggarwal

2014-03-01

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

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

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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

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.

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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.

Characterization of carbon dioxide concentrating chemolithotrophic bacterium Serratia sp. ISTD04 for production of biodiesel.

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Kumar, Manish; Morya, Raj; Gnansounou, Edgard; Larroche, Christian; Thakur, Indu Shekhar

2017-11-01

Proteomics and metabolomics analysis has become a powerful tool for characterization of microbial ability for fixation of Carbon dioxide. Bacterial community of palaeoproterozoic metasediments was enriched in the shake flask culture in the presence of NaHCO 3 . One of the isolate showed resistance to NaHCO 3 (100mM) and was identified as Serratia sp. ISTD04 by 16S rRNA sequence analysis. Carbon dioxide fixing ability of the bacterium was established by carbonic anhydrase enzyme assay along with proteomic analysis by LC-MS/MS. In proteomic analysis 96 proteins were identified out of these 6 protein involved in carbon dioxide fixation, 11 in fatty acid metabolism, indicating the carbon dioxide fixing potency of bacterium along with production of biofuel. GC-MS analysis revealed that hydrocarbons and FAMEs produced by bacteria within the range of C 13 -C 24 and C 11 -C 19 respectively. Presence of 59% saturated and 41% unsaturated organic compounds, make it a better fuel composition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Salivary protein polymorphisms and risk of dental caries: a systematic review.

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Lips, Andrea; Antunes, Leonardo Santos; Antunes, Lívia Azeredo; Pintor, Andrea Vaz Braga; Santos, Diana Amado Baptista Dos; Bachinski, Rober; Küchler, Erika Calvano; Alves, Gutemberg Gomes

2017-06-05

Dental caries is an oral pathology associated with both lifestyle and genetic factors. The caries process can be influenced by salivary composition, which includes ions and proteins. Studies have described associations between salivary protein polymorphisms and dental caries experience, while others have shown no association with salivary proteins genetic variability. The aim of this study is to assess the influence of salivary protein polymorphisms on the risk of dental caries by means of a systematic review of the current literature. An electronic search was performed in PubMed, Scopus, and Virtual Health Library. The following search terms were used: "dental caries susceptibility," "dental caries," "polymorphism, genetics," "saliva," "proteins," and "peptides." Related MeSH headings and free terms were included. The inclusion criteria comprised clinical investigations of subjects with and without caries. After application of these eligibility criteria, the selected articles were qualified by assessing their methodological quality. Initially, 338 articles were identified from the electronic databases after exclusion of duplicates. Exclusion criteria eliminated 322 articles, and 16 remained for evaluation. Eleven articles found a consistent association between salivary protein polymorphisms and risk of dental caries, for proteins related to antimicrobial activity (beta defensin 1 and lysozyme-like protein), pH control (carbonic anhydrase VI), and bacterial colonization/adhesion (lactotransferrin, mucin, and proline-rich protein Db). This systematic review demonstrated an association between genetic polymorphisms and risk of dental caries for most of the salivary proteins.

An olfactory subsystem that detects carbon disulfide and mediates food-related social learning.

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Munger, Steven D; Leinders-Zufall, Trese; McDougall, Lisa M; Cockerham, Renee E; Schmid, Andreas; Wandernoth, Petra; Wennemuth, Gunther; Biel, Martin; Zufall, Frank; Kelliher, Kevin R

2010-08-24

Olfactory signals influence social interactions in a variety of species. In mammals, pheromones and other social cues can promote mating or aggression behaviors; can communicate information about social hierarchies, genetic identity and health status; and can contribute to associative learning. However, the molecular, cellular, and neural mechanisms underlying many olfactory-mediated social interactions remain poorly understood. Here, we report that a specialized olfactory subsystem that includes olfactory sensory neurons (OSNs) expressing the receptor guanylyl cyclase GC-D, the cyclic nucleotide-gated channel subunit CNGA3, and the carbonic anhydrase isoform CAII (GC-D(+) OSNs) is required for the acquisition of socially transmitted food preferences (STFPs) in mice. Using electrophysiological recordings from gene-targeted mice, we show that GC-D(+) OSNs are highly sensitive to the volatile semiochemical carbon disulfide (CS(2)), a component of rodent breath and a known social signal mediating the acquisition of STFPs. Olfactory responses to CS(2) are drastically reduced in mice lacking GC-D, CNGA3, or CAII. Disruption of this sensory transduction cascade also results in a failure to acquire STFPs from either live or surrogate demonstrator mice or to exhibit hippocampal correlates of STFP retrieval. Our findings indicate that GC-D(+) OSNs detect chemosignals that facilitate food-related social interactions. Copyright 2010 Elsevier Ltd. All rights reserved.

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

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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

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

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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

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

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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.

Preclinical validation of 111In-girentuximab-F(ab')2 as a tracer to image hypoxia related marker CAIX expression in head and neck cancer xenografts

NARCIS (Netherlands)

Huizing, F.J.; Hoeben, B.A.W.; Franssen, G.M.; Lok, J.; Heskamp, S.; Oosterwijk, E.; Boerman, O.C.; Bussink, J.

2017-01-01

BACKGROUND AND PURPOSE: Hypoxia is a major cause of radio- and chemoresistance. Carbonic anhydrase IX (CAIX) is an endogenous hypoxia-related marker and an important prognostic marker. Assessment of CAIX expression may allow patient selection for hypoxia or CAIX-targeted treatment. The radioactive

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

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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.

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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.

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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.

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 ...

Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

Science.gov (United States)

Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

2018-06-01

Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24�

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

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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.

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.

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

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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

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.

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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.

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

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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.

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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.

Biochemical characterization of indigenous Fulani and Yoruba ...

African Journals Online (AJOL)

The study was carried out to characterize two indigenous chickens of Nigeria using protein markers; haemolglobin (HB) and carbonic anhydrase (CA). Separation of the two proteins was achieved by cellulose acetate electrophoresis and direct gene counting method was employed to interpret the result. Palentological ...

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

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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

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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

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH.

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Fernández, Pamela A; Hurd, Catriona L; Roleda, Michael Y

2014-12-01

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO3 (-) ) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO3 (-) by the surface-bound enzyme carbonic anhydrase (CAext ). Here, we examined other putative HCO3 (-) uptake mechanisms in M. pyrifera under pHT 9.00 (HCO3 (-) : CO2  = 940:1) and pHT 7.65 (HCO3 (-) : CO2  = 51:1). Rates of photosynthesis, and internal CA (CAint ) and CAext activity were measured following the application of AZ which inhibits CAext , and DIDS which inhibits a different HCO3 (-) uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO3 (-) uptake by M. pyrifera is via an AE protein, regardless of the HCO3 (-) : CO2 ratio, with CAext making little contribution. Inhibiting the AE protein led to a 55%-65% decrease in photosynthetic rates. Inhibiting both the AE protein and CAext at pHT 9.00 led to 80%-100% inhibition of photosynthesis, whereas at pHT 7.65, passive CO2 diffusion supported 33% of photosynthesis. CAint was active at pHT 7.65 and 9.00, and activity was always higher than CAext , because of its role in dehydrating HCO3 (-) to supply CO2 to RuBisCO. Interestingly, the main mechanism of HCO3 (-) uptake in M. pyrifera was different than that in other Laminariales studied (CAext -catalyzed reaction) and we suggest that species-specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO3 (-) :CO2 due to ocean acidification. © 2014 Phycological Society of America.

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.

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.

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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.

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.

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

A Direct Comparison of the MM-GB/SA Scoring Procedure and Free-Energy Perturbation Calculations Using Carbonic Anhydrase as a Test Case: Strengths and Pitfalls of Each Approach.

Science.gov (United States)

Guimarães, Cristiano R W

2011-07-12

MM-GB/SA scoring and free energy perturbation (FEP) calculations have emerged as reliable methodologies to understand structural and energetic relationships to binding. In spite of successful applications to elucidate the structure-activity relationships for few pairs of ligands, the reality is that the performance of FEP calculations has rarely been tested for more than a handful of compounds. In this work, a series of 13 benzene sulfonamide inhibitors of carbonic anhydrase with binding free energies determined by isothermal titration calorimetry was selected as a test case. R(2) values of 0.70, 0.71, and 0.49 with the experiment were obtained with MM-GB/SA and FEP simulations run with MCPRO+ and Desmond, respectively. All methods work well, but the results obtained with Desmond are inferior to MM-GB/SA and MCPRO+. The main contrast between the methods is the level of sampling, ranging from full to restricted flexibility to single conformation for the complexes in Desmond, MCPRO+, and MM-GB/SA, respectively. The current and historical results obtained with MM-GB/SA qualify this approach as a more attractive alternative for rank-ordering; it can achieve equivalent or superior predictive accuracy and handle more structurally dissimilar ligands at a fraction of the computational cost of the rigorous free-energy methods. As for the large theoretical dynamic range for the binding energies, that seems to be a direct result of the degree of sampling in the simulations since MCPRO+ as well as MM-GB/SA are plagued by this. Van't Hoff analysis for selected pairs of ligands suggests that the wider scoring spread is not only affected by missing entropic contributions due to restricted sampling but also exaggerated enthalpic separation between the weak and potent compounds caused by diminished shielding of electrostatic interactions, thermal effects, and protein relaxation/strain.

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

Science.gov (United States)

Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

2018-03-01

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

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.

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

Directory of Open Access Journals (Sweden)

Sasha J. Rose

2016-12-01

Full Text Available Extracellular DNA (eDNA is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM. Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.

The effect of carbonic anhydrase on the kinetics and equilibrium of the oxygen isotope exchange in the CO2-H2O system: Implications for δ18O vital effects in biogenic carbonates

Science.gov (United States)

Uchikawa, Joji; Zeebe, Richard E.

2012-10-01

Interpretations of the primary paleoceanographic information recorded in stable oxygen isotope values (δ18O) of biogenic CaCO3 can be obscured by disequilibrium effects. CaCO3 is often depleted in 18O relative to the δ18O values expected for precipitation in thermodynamic equilibrium with ambient seawater as a result of vital effects. Vital effects in δ18O have been explained in terms of the influence of fluid pH on the overall δ18O of the sum of dissolved inorganic carbon (DIC) species (often referred to as "pH model") and in terms of 18O depletion as a result of the kinetic effects associated with CO2 hydration (CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+) and CO2 hydroxylation (CO2 + OH- ↔ HCO3-) in the calcification sites (so-called "kinetic model"). This study addresses the potential role of an enzyme, carbonic anhydrase (CA), that catalyzes inter-conversion of CO2 and HCO3- in relation to the underlying mechanism of vital effects. We performed quantitative inorganic carbonate precipitation experiments in order to examine the changes in 18O equilibration rate as a function of CA concentration. Experiments were performed at pH 8.3 and 8.9. These pH values are comparable to the average surface ocean pH and elevated pH levels observed in the calcification sites of some coral and foraminiferal species, respectively. The rate of uncatalyzed 18O exchange in the CO2-H2O system is governed by the pH-dependent DIC speciation and the kinetic rate constant for CO2 hydration and hydroxylation, which can be summarized by a simple mathematical expression. The results from control experiments (no CA addition) are in agreement with this expression. The results from control experiments also suggest that the most recently published kinetic rate constant for CO2 hydroxylation has been overestimated. When CA is present, the 18O equilibration process is greatly enhanced at both pH levels due to the catalysis of CO2 hydration by the enzyme. For example, the time required for 18O

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.

A critical analysis of carbonic anhydrase function, respiratory gas exchange, and the acid-base control of secretion in the rectal gland of Squalus acanthias.

Science.gov (United States)

Shuttleworth, Trevor J; Thompson, Jill; Munger, R Stephen; Wood, Chris M

2006-12-01

We compared in vivo responses of rectal gland secretion to carbonic anhydrase (CA) inhibition (10(-4) mol l(-1) acetazolamide) in volume-loaded dogfish with in vitro responses in an isolated-perfused gland stimulated with 5 x 10(-6) mol l(-1) forskolin and removed from systemic influences. We also measured respiratory gas exchange in the perfused gland, described the acid-base status of the secreted fluid, and determined the relative importance of various extracellular and intracellular acid-base parameters in controlling rectal gland secretion in vitro. In vivo, acetazolamide inhibited Cl(-) secretion and decreased pHi in the rectal gland, but interpretation was confounded by an accompanying systemic respiratory acidosis, which would also have contributed to the inhibition. In the perfused gland, M(CO(2)) and M(O(2)) increased in linear relation to increases in Cl(-) secretion rate. CA inhibition (10(-4) mol l(-1) acetazolamide) had no effect on Cl(-) secretion rate or pHi in the perfused gland, in contrast to in vivo, but caused a transitory 30% inhibition of M(CO(2)) (relative to stable M(O(2))) and elevation in secretion P(CO(2)) effects, which peaked at 2 h and attenuated by 3.5-4 h. Secretion was inhibited by acidosis and stimulated by alkalosis; the relationship between relative Cl(-) secretion rate and pHe was almost identical to that seen in vivo. Experimental manipulations of perfusate pH, P(CO(2)) and HCO(3)(-) concentration, together with measurements of pHi, demonstrated that these responses were most strongly correlated with changes in pHe, and were not related to changes in P(CO(2)), extracellular HCO(3)(-), or intracellular HCO(3)(-) levels, though changes in pHi may also have played a role. The acid-base status of the secreted fluid varied with that of the perfusate, secretion pH remaining about 0.3-0.5 units lower, and changing in concert with pHe rather than pHi; secretion HCO(3)(-) concentrations remained low, even in the face of greatly

Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

Science.gov (United States)

Meng Zhang; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

2014-01-01

High throughput screening of protein-small molecule binding interactions using label-free optical biosensors is challenging, as the detected signals are often similar in magnitude to experimental noise. Here, we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with sub-picometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets with binding affinities or inhibition constants in the sub-nanomolar to low micromolar range. The demonstrated ability to perform detection in the presence of several interfering compounds opens the potential for increasing the throughput of the approach. As an example application, we performed a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II (CA II), in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

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.

PENURUNAN KADAR PROTEIN LIMBAH CAIR TAHU DENGAN PEMANFAATAN KARBON BAGASSE TERAKTIVASI (Protein Reduction of Tofu Wastewater Using Activated Carbon Bagasse

Directory of Open Access Journals (Sweden)

Candra Purnawan

2014-10-01

Full Text Available ABSTRAK Penurunan kadar protein limbah tahu telah dilakukan dengan pemanfaatan karbon Bagasse teraktivasi. Tujuan dari penelitian ini adalah untuk mengetahui kondisi optimum dari karbon teraktivasi NaOH dan H2SO4 dalam menurunkan kadar protein limbah cair tahu dan mengetahui jenis isoterm adsorpsi dari karbon aktif yang digunakan untuk menyerap protein limbah cair tahu. Hasil penelitian menunjukkan konsentrasi NaOH yang optimum untuk aktivasi karbon aktif 15%, massa optimum karbon bagasse teraktivasi NaOH adalah 2 g dan penurunan kadar proteinnya 71,95%, sedangkan massa optimum karbon bagasse teraktivasi H2SO4 adalah 1 g dengan penurunan kadar protein sebesar 38,19%. Waktu kontak optimum karbon bagasse teraktivasi  NaOH dan H2SO4 adalah 12 jam. Adsorpsi protein oleh karbon bagasse teraktivasi NaOH mengikuti isoterm adsorpsi Langmuir dan Freundlich sedangkan karbon bagasse teraktivasi H2SO4 dominan mengikuti isoterm Freundlich.   ABSTRACT The protein reduction of tofu wastewater using activated carbon from bagasse  had been conducted. The purposes of this research were to analysis optimum condition of activated carbon bagsse using NaOH and H2SO4 for reduction protein in tofu wastewater, and analysis adsorption isotherm of activated carbon with protein. The result showed that optimum mass of carbon bagasse activated NaOH was  2 g with 71.95% protein reduction, while carbon bagasse activated H2SO4 has 1 g with 38.19% protein reduction. The optimum contact time between protein and activated carbon (with NaOH and H2SO4 was happened in 12 hours. Adsorption protein with carbon bagasse activated NaOH had followed Langmuir and Freundlich adsorption isotherm, while adsorption with carbon bagasse activated H2SO4 dominantlyhad followed Freundlich adsorption isotherm

Measuring binding of protein to gel-bound ligands using magnetic levitation.

Science.gov (United States)

Shapiro, Nathan D; Mirica, Katherine A; Soh, Siowling; Phillips, Scott T; Taran, Olga; Mace, Charles R; Shevkoplyas, Sergey S; Whitesides, George M

2012-03-28

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction-diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems.

Evolution of pH buffers and water homeostasis in eukaryotes: homology between humans and Acanthamoeba proteins.

Science.gov (United States)

Baig, Abdul M; Zohaib, R; Tariq, S; Ahmad, H R

2018-02-01

This study intended to trace the evolution of acid-base buffers and water homeostasis in eukaryotes. Acanthamoeba castellanii  was selected as a model unicellular eukaryote for this purpose. Homologies of proteins involved in pH and water regulatory mechanisms at cellular levels were compared between humans and A. castellanii. Amino acid sequence homology, structural homology, 3D modeling and docking prediction were done to show the extent of similarities between carbonic anhydrase 1 (CA1), aquaporin (AQP), band-3 protein and H + pump. Experimental assays were done with acetazolamide (AZM), brinzolamide and mannitol to observe their effects on the trophozoites of  A. castellanii.  The human CA1, AQP, band-3 protein and H + -transport proteins revealed similar proteins in Acanthamoeba. Docking showed the binding of AZM on amoebal AQP-like proteins.  Acanthamoeba showed transient shape changes and encystation at differential doses of brinzolamide, mannitol and AZM.  Conclusion: Water and pH regulating adapter proteins in Acanthamoeba and humans show significant homology, these mechanisms evolved early in the primitive unicellular eukaryotes and have remained conserved in multicellular eukaryotes.

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.

Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

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Bian, Liujiao; Ji, Xu

2014-01-01

Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio)

DEFF Research Database (Denmark)

Boisen, A M Z; Amstrup, J; Novak, I

2003-01-01

pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA...

Journal of Chemical Sciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Diabatic potential energy surfaces of H + CO .... to locate the minimum energy conformation using quantum chemical theory. ... Effect of electrostatic interactions on the formation of proton transfer pathways in human carbonic anhydrase II ... In silico characterization of antifreeze proteins using computational tools and servers.

The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: structure-function property relationship.

Science.gov (United States)

Depan, D; Misra, R D K

2013-04-01

The rapid adsorption of proteins occurs during the early stages of biomedical device implantation into physiological systems. In this regard, the adsorption of proteins is a strong function of the nature of a biomedical device, which ultimately governs the biological functions. The objective of this study was to elucidate the interplay between nanostructured carbon-modified (graphene oxide and single-walled carbon nanohorn) chitosan scaffolds and consequent protein adsorption and biological function (osteoblast function). We compare and contrast the footprint of protein adsorption on unmodified chitosan and nanostructured carbon-modified chitosan. A comparative analysis of cell-substrate interactions using an osteoblast cell line (MC3T3-E1) implied that biological functions were significantly enhanced in the presence of nanostructured carbon, compared with unmodified chitosan. The difference in their respective behaviors is related to the degree and topography of protein adsorption on the scaffolds. Furthermore, there was a synergistic effect of nanostructured carbon and protein adsorption in terms of favorably modulating biological functions, including cell attachment, proliferation and viability, with the effect being greater on nanostructured carbon-modified scaffolds. The study also underscores that protein adsorption is favored in nanostructured carbon-modified scaffolds such that bioactivity and biological function are promoted. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model.

Directory of Open Access Journals (Sweden)

Maurice Schallenberg

Full Text Available Examination of the response of the retinal proteome to elevated intraocular pressure (IOP and to the pharmacological normalization of IOP is crucial, in order to develop drugs with neuroptorective potential. We used a hereditary rat model of ocular hypertension to lower IOP with travaprost and dorzolamide applied topically on the eye surface, and examine changes of the retinal proteome. Our data demonstrate that elevated IOP causes alterations in the retinal protein profile, in particular in high-mobility-group-protein B1 (HMGB1, calmodulin, heat-shock-protein (HSP 70 and carbonic anhydrase II expression. The changes of the retinal proteome by dorzolamide or travoprost are different and independent of the IOP lowering effect. This fact suggests that the eye drops exert a direct IOP-independent effect on retinal metabolism. Further investigations are required to elucidate the potential neuroprotective mechanisms signaled through changes of HMGB1, calmodulin, HSP70 and carbonic anhydrase II expression in glaucoma. The data may facilitate development of eye drops that exert neuroprotection through direct pharmacological effect.

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.

Carbon source feeding strategies for recombinant protein ...

African Journals Online (AJOL)

USER

2010-04-12

Apr 12, 2010 ... protein expression with the influence of the carbon source feeding ... in the culture media, increasing the peroxisomes numbers ...... source, temperature, pH, O2, methanol feeding strategy) ..... Catabolite Inactivation in Yeast.

Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

Science.gov (United States)

Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J

2016-03-15

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM. Copyright © 2016 the American Physiological Society.

Experimental investigation of interactions between proteins and carbon nanomaterials

Science.gov (United States)

Sengupta, Bishwambhar

The global market for nanomaterials based products is forecasted to reach $1 trillion per annum per annum for 2015. Engineered nanomaterials (ENMs) exhibit unique physicochemical properties with potential to impact diverse aspects of society through applications in electronics, renewable energy, and medicine. While the research and proposed applications of ENMs continue to grow rapidly, the health and safety of ENMs still remains a major concern to the public as well as to policy makers and funding agencies. It is now widely accepted that focused efforts are needed for identifying the list of physicochemical descriptors of ENM before they can be evaluated for nanotoxicity and biological response. This task is surprisingly challenging, as many physicochemical properties of ENMs are closely inter related and cannot be varied independently (e.g. increasing the size of an ENM can introduce additional defects). For example, varying toxic response may ensue due to different methods of nanomaterial preparation, dissimilar impurities and defects. Furthermore, the inadvertent coating of proteins on ENM surface in any biological milieu results in the formation of the so-called "protein/bio-corona" which can in turn alter the fate of ENMs and their biological response. Carbon nanomaterials (CNMs) such as carbon nanotubes, graphene, and graphene oxide are widely used ENMs. It is now known that defects in CNMs play an important role not only in materials properties but also in the determination of how materials interact at the nano-bio interface. In this regard, this work investigates the influence of defect-induced hydrophilicity on the bio-corona formation using micro Raman, photoluminescence, infrared spectroscopy, electrochemistry, and molecular dynamics simulations. Our results show that the interaction of proteins (albumin and fibrinogen) with CNMs is strongly influenced by charge transfer between them, inducing protein unfolding which enhances conformational entropy and

Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

Energy Technology Data Exchange (ETDEWEB)

Song, Y.R. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Wu, B. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Yang, Y.T.; Chen, J. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Zhang, L.J.; Zhang, Z.W. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Shi, H.Y. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Huang, C.L.; Pan, J.X. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Xie, P. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China)

2015-09-08

Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

International Nuclear Information System (INIS)

Song, Y.R.; Wu, B.; Yang, Y.T.; Chen, J.; Zhang, L.J.; Zhang, Z.W.; Shi, H.Y.; Huang, C.L.; Pan, J.X.; Xie, P.

2015-01-01

Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes

Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

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Barwal Indu

2011-12-01

Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

Chemically modified carbon nanotubes as material enhanced laser desorption ionisation (MELDI) material in protein profiling

International Nuclear Information System (INIS)

Najam-ul-Haq, M.; Rainer, M.; Schwarzenauer, T.; Huck, C.W.; Bonn, G.K.

2006-01-01

Biomarkers play a potential role in the early detection and diagnosis of a disease. Our aim is to derivatize carbon nanotubes for exploration of the differences in human body fluids e.g. serum, through matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI/TOF-MS) that can be related to disease and subsequently to be employed in the biomarker discovery process. This application we termed as the material enhanced laser desorption ionisation (MELDI). The versatility of this technology is meant to increase the amount of information from biological samples on the protein level, which will have a major impact to serve the cause of diagnostic markers. Serum peptides and proteins are immobilized on derivatized carbon nanotubes, which function as binding material. Protein-loaded suspension is placed on a stainless steel target or buckypaper on aluminum target for direct analysis with MALDI-MS. The elution method to wash the bound proteins from carbon nanotubes was employed to compare with the direct analysis procedure. Elution is carried out by MALDI matrix solution to get them out of the entangled nanotubes, which are difficult to desorb by laser due to the complex nanotube structures. The advantage of these optimized methods compared to the conventional screening methods is the improved sensitivity, selectivity and the short analysis time without prior albumin and immunoglobulin depletion. The comparison of similarly modified diamond and carbon nanotubes exhibit differences in their nature to bind the proteins out of serum due to the differences in their physical characteristics. Infrared (IR) spectroscopy provided hint for the presence of tertiary amine peak at the crucial chemical step of iminodiacetic acid addition to acid chloride functionality on carbon nanotubes. Atomic absorption spectroscopy (AAS) was utilized to quantitatively measure the copper capacity of these derivatized carbon nanotubes which is a direct measure of capacity of

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

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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

Characterization of the yam tuber storage proteins from Dioscorea batatas exhibiting unique lectin activities.

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Gaidamashvili, Mariam; Ohizumi, Yuki; Iijima, Shinichiro; Takayama, Tomo; Ogawa, Tomohisa; Muramoto, Koji

2004-06-18

Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic

Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity

International Nuclear Information System (INIS)

Zhao, Xingchen; Lu, Dawei; Hao, Fang; Liu, Rutao

2015-01-01

Highlights: • CNT diameter and surface area govern the stability of adsorbed proteins. • More BSA was loaded and destabilized on smaller CNTs. • Protein corona reduces the cytotoxicity of CNTs - Abstract: In this work, we investigated and compared carbon nanotubes (CNTs) of different diameters regarding their interaction with bovine serum albumin (BSA) and their ability to alter protein structure. BSA was exposed to CNT solutions, and the effects were assessed by utilizing fluorescence spectroscopy, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), bichinchoninic acid (BCA) and zeta-potential measurement assays. We demonstrate that CNT diameter and surface area play key roles in influencing the stability of adsorbed proteins. Results showed that the secondary and tertiary structural stability of BSA decreased upon adsorption onto CNTs, with greater decrease on smaller-diametered nanotubes. Besides, more protein was loaded onto CNTs with small diameter, reducing the cytotoxicity. This study, therefore, provides fundamental information for the influence of CNT diameter and surface on protein behavior, which may be helpful to understand toxic effects of CNTs and prove beneficial for developing novel biomedical devices and safe use of nanomaterials

Cluster protein structures using recurrence quantification analysis on coordinates of alpha-carbon atoms of proteins

International Nuclear Information System (INIS)

Zhou Yu; Yu Zuguo; Anh, Vo

2007-01-01

The 3-dimensional coordinates of alpha-carbon atoms of proteins are used to distinguish the protein structural classes based on recurrence quantification analysis (RQA). We consider two independent variables from RQA of coordinates of alpha-carbon atoms, %determ1 and %determ2, which were defined by Webber et al. [C.L. Webber Jr., A. Giuliani, J.P. Zbilut, A. Colosimo, Proteins Struct. Funct. Genet. 44 (2001) 292]. The variable %determ2 is used to define two new variables, %determ2 1 and %determ2 2 . Then three variables %determ1, %determ2 1 and %determ2 2 are used to construct a 3-dimensional variable space. Each protein is represented by a point in this variable space. The points corresponding to proteins from the α, β, α+β and α/β structural classes position into different areas in this variable space. In order to give a quantitative assessment of our clustering on the selected proteins, Fisher's discriminant algorithm is used. Numerical results indicate that the discriminant accuracies are very high and satisfactory

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

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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.

Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization

Energy Technology Data Exchange (ETDEWEB)

Singh, Shiv; Singh, Abhinav [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Bais, Vaibhav Sushil Singh; Prakash, Balaji [Department of Biological Science and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Verma, Nishith, E-mail: nishith@iitk.ac.in [Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Center for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

2014-05-01

In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultra-sonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, pore-size distribution, and UV–vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules. - Highlights: • Ni metal Np-dispersed carbon micro-nanofibers (ACFs/CNFs) are prepared. • ACFs/CNFs are mesoporous. • Significant adsorption of BSA, GOx and YqeH is observed on ACFs/CNFs. • Multi-scale web of ACFs/CNFs is effective for protein immobilization.

Protein mapping of calcium carbonate biominerals by immunogold.

Science.gov (United States)

Marin, Frédéric; Pokroy, Boaz; Luquet, Gilles; Layrolle, Pierre; De Groot, Klaas

2007-05-01

The construction of metazoan calcium carbonate skeletons is finely regulated by a proteinaceous extracellular matrix, which remains embedded within the exoskeleton. In spite of numerous biochemical studies, the precise localization of skeletal proteins has remained for a long time as an elusive goal. In this paper, we describe a technique for visualizing shell matrix proteins on the surface of calcium carbonate crystals or within the biominerals. The technique is as follows: freshly broken pieces of biominerals or NaOCl then EDTA-etched polished surfaces are incubated with an antibody elicited against one matrix protein, then with a secondary gold-coupled antibody. After silver enhancement, the samples are subsequently observed with scanning electron microscopy by using back-scattered electron mode. In the present case, the technique is applied to a particular example, the calcitic prisms that compose the outer shell layer of the mediterranean fan mussel Pinna nobilis. One major soluble protein, caspartin, which was identified recently, was partly de novo sequenced after enzymatic digestions. A polyclonal antibody raised against caspartin was used for its localization within and on the prisms. The immunogold localization indicated that caspartin surrounds the calcitic prisms, but is also dispersed within the biominerals. This example illustrates the deep impact of the technique on the definition of intracrystalline versus intercrystalline matrix proteins. Furthermore, it is an important tool for assigning a putative function to a matrix protein of interest.

Magnetic levitation as a platform for competitive protein-ligand binding assays.

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Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

2012-07-17

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

Evaluation of protein immobilization capacity on various carbon nanotube embedded hydrogel biomaterials

Energy Technology Data Exchange (ETDEWEB)

Derkus, Burak, E-mail: burakderkus@gmail.com; Emregul, Kaan Cebesoy; Emregul, Emel

2015-11-01

This study investigates effective immobilization of proteins, an important procedure in many fields of bioengineering and medicine, using various biomaterials. Gelatin, alginate and chitosan were chosen as polymeric carriers, and applied in both their composites and nanocomposite forms in combination with carbon nanotubes (CNTs). The prepared nano/composite structures were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and contact angle analysis (CA). Electrochemical impedance spectroscopy analysis revealed gelatin composites in general to exhibit better immobilization performance relative to the native gelatin which can be attributed to enhanced film morphologies of the composite structures. Moreover, superior immobilization efficiencies were obtained with the addition of carbon nanotubes, due to their conducting and surface enhancement features, especially in the gelatin–chitosan structures due to the presence of structural active groups. - Highlights: • Various nanocomposite biomaterials were developed for efficient immobilization of proteins. • CNTs enhance the immobilization efficiency owing to their conducting and surface enhancement features. • Gelatin–chitosan–CNTs structure is promising immobilization matrix thanks to its effective CNTs binding capacity.

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

Science.gov (United States)

Sharick, Joe T; Favreau, Peter F; Gillette, Amani A; Sdao, Sophia M; Merrins, Matthew J; Skala, Melissa C

2018-04-03

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ 2 ) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ 2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization.

Science.gov (United States)

Sanchez, Sophie; Hourdez, Stéphane; Lallier, François H

2007-09-24

Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Quantitative PCR analyses were congruent with our libraries

Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization

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Lallier François H

2007-09-01

Full Text Available Abstract Background Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing γ-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water and in the trophosome (the organ housing the symbiotic bacteria using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species. Results We produced four cDNA libraries: i body wall-subtracted branchial plume library (BR-BW, ii and its reverse library, branchial plume-subtracted body wall library (BW-BR, iii body wall-subtracted trophosome library (TR-BW, iv and its reverse library, trophosome-subtracted body wall library (BW-TR. For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively. Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr, cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr, myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue. Conclusion Quantitative PCR

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii.

Directory of Open Access Journals (Sweden)

João Vitor Dutra Molino

Full Text Available Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

Clustering of two genes putatively involved in cyanate detoxification evolved recently and independently in multiple fungal lineages

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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...

Carbon Nanotubes Facilitate Oxidation of Cysteine Residues of Proteins.

Science.gov (United States)

Hirano, Atsushi; Kameda, Tomoshi; Wada, Momoyo; Tanaka, Takeshi; Kataura, Hiromichi

2017-10-19

The adsorption of proteins onto nanoparticles such as carbon nanotubes (CNTs) governs the early stages of nanoparticle uptake into biological systems. Previous studies regarding these adsorption processes have primarily focused on the physical interactions between proteins and nanoparticles. In this study, using reduced lysozyme and intact human serum albumin in aqueous solutions, we demonstrated that CNTs interact chemically with proteins. The CNTs induce the oxidation of cysteine residues of the proteins, which is accounted for by charge transfer from the sulfhydryl groups of the cysteine residues to the CNTs. The redox reaction simultaneously suppresses the intermolecular association of proteins via disulfide bonds. These results suggest that CNTs can affect the folding and oxidation degree of proteins in biological systems such as blood and cytosol.

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.

Effects of prolonged recombinant human erythropoietin administration on muscle membrane transport systems and metabolic marker enzymes

DEFF Research Database (Denmark)

Juel, C; Thomsen, J J; Rentsch, R L

2007-01-01

on the expression of muscle membrane transport proteins. Likewise, improvements in performance may involve upregulation of metabolic enzymes. Since Epo is known to augment performance we tested the effect of rHuEpo on some marker enzymes that are related to aerobic capacity. For these purposes eight subjects...... performance by approximately 54%. Membrane transport systems and carbonic anhydrases involved in pH regulation remained unchanged. Of the Na(+), K(+)-pump isoforms only the density of the alpha2 subunit was decreased (by 22%) after treatment. The marker enzymes cytochrom c and hexokinase remained unchanged......Adaptations to chronic hypoxia involve changes in membrane transport proteins. The underlying mechanism of this response may be related to concomitant occurring changes in erythropoietin (Epo) levels. We therefore tested the direct effects of recombinant human erythropoietin (rHuEpo) treatment...

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

Lipid-rich and protein-poor carbon allocation patterns of phytoplankton in the northern Chukchi Sea, 2011

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Yun, Mi Sun; Joo, Hui Tae; Park, Jung Woo; Kang, Jae Joong; Kang, Sung-Ho; Lee, Sang H.

2018-04-01

The carbon allocations of phytoplankton into different photosynthetic end products (lipids, LMWM, polysaccharides, and proteins) were determined to understand physiological conditions of phytoplankton in the northern Chukchi Sea during the Korean Arctic expedition, 2011, using the 13C isotope tracer technique. The carbon allocation rates of lipids, LMWM, polysaccharides, and proteins were 0.00009-0.00062 h-1, 0.00001-0.00049 h-1, 0.00001-0.00025 h-1, and 0.00001-0.00062 h-1 within the euphotic depths from surface to 1% light depths during our cruise period, respectively. Significant relationships between protein production rates and chlorophyll a concentrations (large and total) were found in this study. Moreover, we found a significant negative relationship between lipid production rates and ammonium concentrations. These relationships match well with the previous results for environmental/physiological conditions for phytoplankton growth. Overall, phytoplankton allocated more photosynthetic carbon into lipids (42.5 ± 17.7%) whereas relatively lower to proteins (20.4 ± 15.5%) in this study. The lipid-rich and protein-poor allocation patterns in this study suggest that phytoplankton in the northern Chukchi Sea were in a stationary growth phase under nutrient deficient condition based on biological and environmental conditions observed during our study period. Based on comparison with the previous studies in the northern Bering Sea and southern Chukchi Sea, we found that the photosynthetic carbon allocation patterns depending on physiological status of phytoplankton under the different growth and/or nutrient conditions could be largely vary at different regions in the Arctic Ocean. More intensive research on the physiological status of phytoplankton is further required to determine how phytoplankton response to the changing environmental conditions and consequently how they impact on higher trophic levels in marine ecosystems in the Arctic Ocean.

CRP-Mediated Carbon Catabolite Regulation of Yersinia pestis Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA.

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Stephan P Willias

Full Text Available The natural transmission of Yersinia pestis is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate Y. pestis biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon Y. pestis biofilm production. Our findings demonstrate that Y. pestis biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of crp in both CO92 and KIM6+ Y. pestis strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter Y. pestis biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized hms biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote Y. pestis biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon Y. pestis biofilm production. Contrary to what has been described for E. coli, Y. pestis biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of csrA significantly impaired Y. pestis biofilm production. CsrA was found to promote Y. pestis biofilm production independent of glycogen regulation. Loss of csrA did not significantly alter relative hmsH, hmsP, or hmsT mRNA abundance. However, deletion of hmsP in the csrA-deficient mutant enabled excessive biofilm production

Endosialin and Associated Protein Expression in Soft Tissue Sarcomas: A Potential Target for Anti-Endosialin Therapeutic Strategies

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Daniel J. O’Shannessy

2016-01-01

Full Text Available Endosialin (CD248, TEM-1 is expressed in pericytes, tumor vasculature, tumor fibroblasts, and some tumor cells, including sarcomas, with limited normal tissue expression, and appears to play a key role in tumor-stromal interactions, including angiogenesis. Monoclonal antibodies targeting endosialin have entered clinical trials, including soft tissue sarcomas. We evaluated a cohort of 94 soft tissue sarcoma samples to assess the correlation between gene expression and protein expression by immunohistochemistry for endosialin and PDGFR-β, a reported interacting protein, across available diagnoses. Correlations between the expression of endosialin and 13 other genes of interest were also examined. Within cohorts of soft tissue diagnoses assembled by tissue type (liposarcoma, leiomyosarcoma, undifferentiated sarcoma, and other, endosialin expression was significantly correlated with a better outcome. Endosialin expression was highest in liposarcomas and lowest in leiomyosarcomas. A robust correlation between protein and gene expression data for both endosialin and PDGFR-β was observed. Endosialin expression positively correlated with PDGFR-β and heparin sulphate proteoglycan 2 and negatively correlated with carbonic anhydrase IX. Endosialin likely interacts with a network of extracellular and hypoxia activated proteins in sarcomas and other tumor types. Since expression does vary across histologic groups, endosialin may represent a selective target in soft tissue sarcomas.

Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

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Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

2017-10-01

Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

Carbon dioxide capture using resin-wafer electrodeionization

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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.

Mesoporous silica particles modified with graphitic carbon: interaction with human red blood cells and plasma proteins

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Martinez, Diego Stefani Teodoro; Franqui, Lidiane Silva; Bettini, Jefferson; Strauss, Mathias, E-mail: diego.martinez@lnnano.cnpem.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Damasceno, Joao Paulo Vita; Mazali, Italo Odone [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

2016-07-01

Full text: In this work the interaction of the mesoporous silica particles (SBA-15, ∼700 nm) modified with graphitic carbon (SBA-15/C) on human red blood cells (hemolysis) and plasma proteins (protein corona formation) is studied. XPS and CHN analysis showed that the carbon content on the SBA-15/C samples varied from 2 to 10% and was tuned by the functionalization step. The formed carbon structures where associated to graphitic nanodomains coating the pores surface as verified by Raman spectroscopy and {sup 13}C NMR. Advanced TEM/EELS analysis showed that the carbon structures are distributed along the SBA-15 mesopores. SAXS and textural analyses were used to confirm that the porous structure of the silica support is kept after the modification procedure and to calculate the number of graphitic carbon stacked layers coating the mesopores. After incubation of SBA-15 with human red blood cells (RBCs), it was observed a dose-dependent hemolytic effect, probably, due to binding of the material silanol-rich surface to the phosphatidylcholine molecules from the RBC membrane. The graphitic carbon modifications have mitigated this effect, indicating that the graphitic carbon coating protected the silanol groups of the particle surface hindering the hemolysis. Considering the protein corona formation, selective biomolecular interaction of proteins was observed for the different materials using gel electrophoresis (SDS-PAGE) analysis. Besides, graphitic carbon modification decreased the amount of proteins on the corona. Together, the in vitro hemolysis and protein corona assays are promising biological models to understand the influence of silica surface functionalization on their bionano-interactions. Finally, our work contributes to the development of fundamental research on such nanomaterials chemistry in the emerging field of nanobioscience and nanotoxicology. (author)

Proteomic Identification of Target Proteins of Thiodigalactoside in White Adipose Tissue from Diet-Induced Obese Rats

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Hilal Ahmad Parray

2015-06-01

Full Text Available Previously, galectin-1 (GAL1 was found to be up-regulated in obesity-prone subjects, suggesting that use of a GAL1 inhibitor could be a novel therapeutic approach for treatment of obesity. We evaluated thiodigalactoside (TDG as a potent inhibitor of GAL1 and identified target proteins of TDG by performing comparative proteome analysis of white adipose tissue (WAT from control and TDG-treated rats fed a high fat diet (HFD using two dimensional gel electrophoresis (2-DE combined with MALDI-TOF-MS. Thirty-two spots from a total of 356 matched spots showed differential expression between control and TDG-treated rats, as identified by peptide mass fingerprinting. These proteins were categorized into groups such as carbohydrate metabolism, tricarboxylic acid (TCA cycle, signal transduction, cytoskeletal, and mitochondrial proteins based on functional analysis using Protein Annotation Through Evolutionary Relationship (PANTHER and Database for Annotation, Visualization, Integrated Discovery (DAVID classification. One of the most striking findings of this study was significant changes in Carbonic anhydrase 3 (CA3, Voltage-dependent anion channel 1 (VDAC1, phosphatidylethanolamine-binding protein 1 (PEBP1, annexin A2 (ANXA2 and lactate dehydrogenase A chain (LDHA protein levels between WAT from control and TDG-treated groups. In addition, we confirmed increased expression of thermogenic proteins as well as reduced expression of lipogenic proteins in response to TDG treatment. These results suggest that TDG may effectively prevent obesity, and TDG-responsive proteins can be used as novel target proteins for obesity treatment.

Plasma protein profiling of patients with intraductal papillary mucinous neoplasm of the pancreas as potential precursor lesions of pancreatic cancer.

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Ilies, Maria; Sappa, Praveen Kumar; Iuga, Cristina Adela; Loghin, Felicia; Gesell Salazar, Manuela; Weiss, Frank Ulrich; Beyer, Georg; Lerch, Markus M; Völker, Uwe; Mayerle, Julia; Hammer, Elke

2018-02-01

Efforts for the early diagnosis of the pancreatic ductal adenocarcinoma (PDAC) have recently been driven to one of the precursor lesions, namely intraductal papillary mucinous neoplasm of the pancreas (IPMN). Only a few studies have focused on IPMN molecular biology and its overall progression to cancer. Therefore, IPMN lacks comprehensive characterization which makes its clinical management controversial. In this study, we characterized plasma proteins in the presence of IPMNs in comparison to healthy controls, chronic pancreatitis, and PDAC by a proteomics approach using data-independent acquisition based mass spectrometry. We describe several protein sets that could aid IPMN diagnosis, but also differentiation of IPMN from healthy controls, as well as from benign and malignant diseases. Among all, high levels of carbonic anhydrases and hemoglobins were characteristic for the IPMN group. By employing ELISA based quantification we validated our results for human tissue inhibitor of metalloproteinase inhibitor 1 (TIMP-1). We consider IPMN management directed towards an early potential cancer development a crucial opportunity before PDAC initiation and thus its early detection and cure. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure and function of complex I in animals and plants - a comparative view.

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Senkler, Jennifer; Senkler, Michael; Braun, Hans-Peter

2017-09-01

The mitochondrial NADH dehydrogenase complex (complex I) has a molecular mass of about 1000 kDa and includes 40-50 subunits in animals, fungi and plants. It is composed of a membrane arm and a peripheral arm and has a conserved L-like shape in all species investigated. However, in plants and possibly some protists it has a second peripheral domain which is attached to the membrane arm on its matrix exposed side at a central position. The extra domain includes proteins resembling prokaryotic gamma-type carbonic anhydrases. We here present a detailed comparison of complex I from mammals and flowering plants. Forty homologous subunits are present in complex I of both groups of species. In addition, five subunits are present in mammalian complex I, which are absent in plants, and eight to nine subunits are present in plant complex I which do not occur in mammals. Based on the atomic structure of mammalian complex I and biochemical insights into complex I architecture from plants we mapped the species-specific subunits. Interestingly, four of the five animal-specific and five of the eight to nine plant-specific subunits are localized at the inner surface of the membrane arm of complex I in close proximity. We propose that the inner surface of the membrane arm represents a workbench for attaching proteins to complex I, which are not directly related to respiratory electron transport, like nucleoside kinases, acyl-carrier proteins or carbonic anhydrases. We speculate that further enzyme activities might be bound to this micro-location in other groups of organisms. © 2017 Scandinavian Plant Physiology Society.

Proteins involved in flor yeast carbon metabolism under biofilm formation conditions.

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Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Mauricio, Juan Carlos

2015-04-01

A lack of sugars during the production of biologically aged wines after fermentation of grape must causes flor yeasts to metabolize other carbon molecules formed during fermentation (ethanol and glycerol, mainly). In this work, a proteome analysis involving OFFGEL fractionation prior to LC/MS detection was used to elucidate the carbon metabolism of a flor yeast strain under biofilm formation conditions (BFC). The results were compared with those obtained under non-biofilm formation conditions (NBFC). Proteins associated to processes such as non-fermentable carbon uptake, the glyoxylate and TCA cycles, cellular respiration and inositol metabolism were detected at higher concentrations under BFC than under the reference conditions (NBFC). This study constitutes the first attempt at identifying the flor yeast proteins responsible for the peculiar sensory profile of biologically aged wines. A better metabolic knowledge of flor yeasts might facilitate the development of effective strategies for improved production of these special wines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Carbon Nanotubes: A Review on Structure and Their Interaction with Proteins

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N. Saifuddin

2013-01-01

Full Text Available Carbon nanotubes (CNTs are allotropes of carbon with a nanostructure that can have a length-to-diameter ratio greater than 1,000,000. Techniques have been developed to produce nanotubes in sizeable quantities, including arc discharge, laser ablation, and chemical vapor deposition. Developments in the past few years have illustrated the potentially revolutionizing impact of nanomaterials, especially in biomedical imaging, drug delivery, biosensing, and the design of functional nanocomposites. Methods to effectively interface proteins with nanomaterials for realizing these applications continue to evolve. The high surface-to-volume ratio offered by nanoparticles resulted in the concentration of the immobilized entity being considerably higher than that afforded by other materials. There has also been an increasing interest in understanding the influence of nanomaterials on the structure and function of proteins. Various immobilization methods have been developed, and in particular, specific attachment of enzymes on carbon nanotubes has been an important focus of attention. With the growing attention paid to cascade enzymatic reaction, it is possible that multienzyme coimmobilization would be one of the next goals in the future. In this paper, we focus on advances in methodology for enzyme immobilization on carbon nanotubes.

Evaluation of protein immobilization capacity on various carbon nanotube embedded hydrogel biomaterials.

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Derkus, Burak; Emregul, Kaan Cebesoy; Emregul, Emel

2015-11-01

This study investigates effective immobilization of proteins, an important procedure in many fields of bioengineering and medicine, using various biomaterials. Gelatin, alginate and chitosan were chosen as polymeric carriers, and applied in both their composites and nanocomposite forms in combination with carbon nanotubes (CNTs). The prepared nano/composite structures were characterized using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TG) and contact angle analysis (CA). Electrochemical impedance spectroscopy analysis revealed gelatin composites in general to exhibit better immobilization performance relative to the native gelatin which can be attributed to enhanced film morphologies of the composite structures. Moreover, superior immobilization efficiencies were obtained with the addition of carbon nanotubes, due to their conducting and surface enhancement features, especially in the gelatin-chitosan structures due to the presence of structural active groups. Copyright © 2015 Elsevier B.V. All rights reserved.

Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

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Aayudh Das

2016-01-01

Full Text Available Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

Ultra-Thin Optically Transparent Carbon Electrodes Produced from Layers of Adsorbed Proteins

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Alharthi, Sarah A.; Benavidez, Tomas E.; Garcia, Carlos D.

2013-01-01

This work describes a simple, versatile, and inexpensive procedure to prepare optically transparent carbon electrodes, using proteins as precursors. Upon adsorption, the protein-coated substrates were pyrolyzed under reductive conditions (5% H2) to form ultra-thin, conductive electrodes. Because proteins spontaneously adsorb to interfaces forming uniform layers, the proposed method does not require a precise control of the preparation conditions, specialized instrumentation, or expensive precursors. The resulting electrodes were characterized by a combination of electrochemical, optical, and spectroscopic means. As a proof-of-concept, the optically-transparent electrodes were also used as substrate for the development of an electrochemical glucose biosensor. The proposed films represent a convenient alternative to more sophisticated, and less available, carbon-based nanomaterials. Furthermore, these films could be formed on a variety of substrates, without classical limitations of size or shape. PMID:23421732

Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

Energy Technology Data Exchange (ETDEWEB)

Yu, Yuehua; Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Shin, Yongsoon; Lei, Chenghong; Liu, Jun

2012-05-01

We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can be released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.

Ocean acidification impacts mussel control on biomineralisation.

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Fitzer, Susan C; Phoenix, Vernon R; Cusack, Maggie; Kamenos, Nicholas A

2014-08-28

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.

Biological conversion of carbon dioxide and hydrogen into liquid fuels and industrial chemicals.

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Hawkins, Aaron S; McTernan, Patrick M; Lian, Hong; Kelly, Robert M; Adams, Michael W W

2013-06-01

Non-photosynthetic routes for biological fixation of carbon dioxide into valuable industrial chemical precursors and fuels are moving from concept to reality. The development of 'electrofuel'-producing microorganisms leverages techniques in synthetic biology, genetic and metabolic engineering, as well as systems-level multi-omic analysis, directed evolution, and in silico modeling. Electrofuel processes are being developed for a range of microorganisms and energy sources (e.g. hydrogen, formate, electricity) to produce a variety of target molecules (e.g. alcohols, terpenes, alkenes). This review examines the current landscape of electrofuel projects with a focus on hydrogen-utilizing organisms covering the biochemistry of hydrogenases and carbonic anhydrases, kinetic and energetic analyses of the known carbon fixation pathways, and the state of genetic systems for current and prospective electrofuel-producing microorganisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

Close Association of Carbonic Anhydrase (CA2a and CA15a), Na+/H+ Exchanger (Nhe3b), and Ammonia Transporter Rhcg1 in Zebrafish Ionocytes Responsible for Na+ Uptake

Science.gov (United States)

Ito, Yusuke; Kobayashi, Sayako; Nakamura, Nobuhiro; Miyagi, Hisako; Esaki, Masahiro; Hoshijima, Kazuyuki; Hirose, Shigehisa

2013-01-01

Freshwater (FW) fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were upregulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morpholino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+uptake under FW conditions by forming a transport metabolon with Nhe3b. PMID:23565095

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

Science.gov (United States)

Quinn, Patrick; Bowers, Robert M; Zhang, Xiaoyu; Wahlund, Thomas M; Fanelli, Michael A; Olszova, Daniela; Read, Betsy A

2006-08-01

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

Prioritizing Popular Proteins in Liver Cancer: Remodelling One-Carbon Metabolism.

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Mora, María Isabel; Molina, Manuela; Odriozola, Leticia; Elortza, Félix; Mato, José María; Sitek, Barbara; Zhang, Pumin; He, Fuchu; Latasa, María Uxue; Ávila, Matías Antonio; Corrales, Fernando José

2017-12-01

Primary liver cancer (HCC) is recognized as the fifth most common neoplasm and the second leading cause of cancer death worldwide. Most risk factors are known, and the molecular pathogenesis has been widely studied in the past decade; however, the underlying molecular mechanisms remain to be unveiled, as they will facilitate the definition of novel biomarkers and clinical targets for more effective patient management. We utilize the B/D-HPP popular protein strategy. We report a list of popular proteins that have been highly cocited with the expression "liver cancer". Several enzymes highlight the known metabolic remodeling of liver cancer cells, four of which participate in one-carbon metabolism. This pathway is central to the maintenance of differentiated hepatocytes, as it is considered the connection between intermediate metabolism and epigenetic regulation. We designed a targeted selective reaction monitoring (SRM) method to follow up one-carbon metabolism adaptation in mouse HCC and in regenerating liver following exposure to CCl 4 . This method allows systematic monitoring of one-carbon metabolism and could prove useful in the follow-up of HCC and of chronically liver-diseased patients (cirrhosis) at risk of HCC. The SRM data are available via ProteomeXchange in PASSEL (PASS01060).

Preclinical evaluation of carbon-11 and fluorine-18 sulfonamide derivatives for in vivo radiolabeling of erythrocytes

OpenAIRE

Gheysens, Olivier; Akurathi, Vamsidhar; Chekol, Rufael; Dresselaers, Tom; Celen, Sofie; Koole, Michel; Dauwe, Dieter; Cleynhens, Bernard J; Claus, Piet; Janssens, Stefan; Verbruggen, Alfons M; Nuyts, Johan; Himmelreich, Uwe; Bormans, Guy M

2013-01-01

Background To date, few PET tracers for in vivo labeling of red blood cells (RBCs) are available. In this study, we report the radiosynthesis and in vitro and in vivo evaluation of 11C and 18F sulfonamide derivatives targeting carbonic anhydrase II (CA II), a metallo-enzyme expressed in RBCs, as potential blood pool tracers. A proof-of-concept in vivo imaging study was performed to demonstrate the feasibility to assess cardiac function and volumes using electrocardiogram (ECG)-gated positron ...

Electrochemical immunosensors for the detection of survival motor neuron (SMN) protein using different carbon nanomaterials-modified electrodes.

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Eissa, Shimaa; Alshehri, Nawal; Rahman, Anas M Abdel; Dasouki, Majed; Abu-Salah, Khalid M; Zourob, Mohammed

2018-03-15

Spinal muscular atrophy is an untreatable potentially fatal hereditary disorder caused by loss-of-function mutations in the survival motor neuron (SMN) 1 gene which encodes the SMN protein. Currently, definitive diagnosis relies on the demonstration of biallelic pathogenic variants in SMN1 gene. Therefore, there is an urgent unmet need to accurately quantify SMN protein levels for screening and therapeutic monitoring of symptomatic newborn and SMA patients, respectively. Here, we developed a voltammetric immunosensor for the sensitive detection of SMN protein based on covalently functionalized carbon nanofiber-modified screen printed electrodes. A comparative study of six different carbon nanomaterial-modified electrodes (carbon, graphene (G), graphene oxide (GO), single wall carbon nanotube (SWCNT), multi-wall carbon nanotube (MWCNT), and carbon nanofiber (CNF)) was performed. 4-carboxyphenyl layers were covalently grafted on the six electrodes by electroreduction of diazonium salt. Then, the terminal carboxylic moieties on the electrodes surfaces were utilized to immobilize the SMN antibody via EDC/NHS chemistry and to fabricate the immunosensors. The electrochemical characterization and analytical performance of the six immunosensors suggest that carbon nanofiber is a better electrode material for the SMN immunosensor. The voltammetric SMN carbon nanofiber-based immunosensor showed high sensitivity (detection limit of 0.75pg/ml) and selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and dystrophin (DMD). We suggest that this novel biosensor is superior to other developed assays for SMN detection in terms of lower cost, higher sensitivity, simplicity and capability of high throughput screening. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

Science.gov (United States)

Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

2016-01-01

Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa ( Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

Optically and biologically active mussel protein-coated double-walled carbon nanotubes.

Science.gov (United States)

Jung, Yong Chae; Muramatsu, Hiroyuki; Fujisawa, Kazunori; Kim, Jin Hee; Hayashi, Takuya; Kim, Yoong Ahm; Endo, Morinobu; Terrones, Mauricio; Dresselhaus, Mildred S

2011-12-02

A method of dispersing strongly bundled double-walled carbon nanotubes (DWNTs) via a homogeneous coating of mussel protein in an aqueous solution is presented. Optical activity, mechanical strength, as well as electrical conductivity coming from the nanotubes and the versatile biological activity from the mussel protein make mussel-coated DWNTs promising as a multifunctional scaffold and for anti-fouling materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High incorporation of carbon into proteins by the phytoplankton of the Bering Strait and Chukchi Sea

Science.gov (United States)

Lee, Sang H.; Kim, Hak-Jun; Whitledge, Terry E.

2009-07-01

High incorporation of carbon into proteins and low incorporation into lipids were a characteristic pattern of the photosynthetic allocations of phytoplankton throughout the euphotic zone in the Bering Strait and Chukchi Sea in 2004. According to earlier studies, this indicates that phytoplankton had no nitrogen limitation and a physiologically healthy condition, at least during the cruise period from mid-August to early September in 2004. This is an interesting result, especially for the phytoplankton in the Alaskan coastal water mass-dominated region in the Chukchi Sea which has been thought to be potentially nitrogen limited. The relatively high ammonium concentration is believed to have supported the nitrogen demand of the phytoplankton in the region where small cells (stress than large phytoplankton. If the high carbon incorporation into proteins by the phytoplankton in 2004 is a general pattern of the photosynthetic allocations in the Chukchi Sea, they could provide nitrogen-sufficient food for the highest benthic faunal biomass in the Arctic Ocean, sustaining large populations of benthic-feeding marine mammals and seabirds.

Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

International Nuclear Information System (INIS)

Singh, Renu; Hong, Seongkyeol; Jang, Jaesung

2015-01-01

Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO 2 aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO 2 ) aerosols (a mixture of solid and gaseous CO 2 ), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL −1 ) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO 2 aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors

Parathyroid hormone-related protein blood test

Science.gov (United States)

... ency/article/003691.htm Parathyroid hormone-related protein blood test To use the sharing features on this page, ... measures the level of a hormone in the blood, called parathyroid hormone-related protein. How the Test is Performed A blood sample is needed . How ...

Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

Directory of Open Access Journals (Sweden)

Carol B Fowler

2010-12-01

Full Text Available Proteomic studies of formalin-fixed paraffin-embedded (FFPE tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%. Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

Science.gov (United States)

Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

2010-01-01

Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

Optic nerve oxygen tension

DEFF Research Database (Denmark)

la Cour, M; Kiilgaard, Jens Folke; Eysteinsson, T

2000-01-01

To investigate the influence of acute changes in intraocular pressure on the oxygen tension in the vicinity of the optic nerve head under control conditions and after intravenous administration of 500 mg of the carbonic anhydrase inhibitor dorzolamide.......To investigate the influence of acute changes in intraocular pressure on the oxygen tension in the vicinity of the optic nerve head under control conditions and after intravenous administration of 500 mg of the carbonic anhydrase inhibitor dorzolamide....

Ocean acidification modulates expression of genes and physiological performance of a marine diatom

Science.gov (United States)

Li, Y.; Zhuang, S.; Wu, Y.; Ren, H.; Cheng, F.; Lin, X.; Wang, K.; Beardall, J.; Gao, K.

2015-09-01

Ocean Acidification (OA) is known to affect various aspects of the physiological performance of diatoms, but there is little information on the underlining molecular mechanisms involved. Here, we show that in the model diatom Phaeodactylum tricornutum expression of the genes related to light harvesting, carbon acquisition and carboxylation, nitrite assimilation and ATP synthesis are modulated by OA. Growth and photosynthetic carbon fixation were enhanced by elevated CO2 (1000 μatm) under both constant indoor and fluctuating outdoor light regimes. The genetic expression of nitrite reductase (NiR) was up-regulated by OA regardless of light levels and/or regimes. The transcriptional expression of fucoxanthin chlorophyll a/c protein (lhcf type (FCP)) and mitochondrial ATP synthase (mtATP synthase) genes were also enhanced by OA, but only under high light intensity. OA treatment decreased the expression of β-carbonic anhydrase (β-CA) along with down-regulation of CO2 concentrating mechanisms (CCMs). Additionally, the genes for these proteins (NiR, FCP, mtATP synthase, β-CA) showed diel expressions either under constant indoor light or fluctuating sunlight. Thus, OA enhanced photosynthetic and growth rates by stimulating nitrogen assimilation and indirectly by down-regulating the energy-costly inorganic carbon acquisition process.

Acetazolamide on the ventral medulla of the cat increases phrenic output and delays the ventilatory response to CO sub 2

Energy Technology Data Exchange (ETDEWEB)

Coates, E.L.; Aihua Li; Nattie, E.E. (Dartmouth College, Hanover, NH (United States))

1991-03-11

Acetazolamide applied to the surface of the rostral ventrolateral medulla or microinjected beneath the medullary surface in chloralose-urethan-anesthetized, vagotomized, carotid denervated, paralyzed, servo-ventilated cats produced a long-lasting increase in phrenic minute ventilation. Extracellular pH measured beneath the rostral ventrolateral medulla exhibited a long-lasting decrease after surface acetazolamide but was not a good predictor, in each individual animal, of changes in phrenic activity. Medullary carbonic anhydrase inhibition reduced the slope and the half-time of the phrenic response to rapid step CO{sub 2} increases. Conversely, acetazolamide did not effect the phrenic response to steady state CO{sub 2} increases. These data indicate that localized inhibition of medullary carbonic anhydrase causes a centrally mediated increase in ventilation that the authors attribute to medullary tissue acidosis. In addition, these data indicate that medullary carbonic anhydrase may play a role in central CO{sub 2} chemotransduction.

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

Directory of Open Access Journals (Sweden)

Marilda Rigobello-Masini

2003-07-01

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

Nitrate Activation of Cytosolic Protein Kinases Diverts Photosynthetic Carbon from Sucrose to Amino Acid Biosynthesis

Science.gov (United States)

Champigny, Marie-Louise; Foyer, Christine

1992-01-01

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis. PMID:16653003

Protein function prediction using neighbor relativity in protein-protein interaction network.

Science.gov (United States)

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

Exploring possible mechanisms of action for the nanotoxicity and protein binding of decorated nanotubes: interpretation of physicochemical properties from optimal QSAR models.

Science.gov (United States)

Esposito, Emilio Xavier; Hopfinger, Anton J; Shao, Chi-Yu; Su, Bo-Han; Chen, Sing-Zuo; Tseng, Yufeng Jane

2015-10-01

Carbon nanotubes have become widely used in a variety of applications including biosensors and drug carriers. Therefore, the issue of carbon nanotube toxicity is increasingly an area of focus and concern. While previous studies have focused on the gross mechanisms of action relating to nanomaterials interacting with biological entities, this study proposes detailed mechanisms of action, relating to nanotoxicity, for a series of decorated (functionalized) carbon nanotube complexes based on previously reported QSAR models. Possible mechanisms of nanotoxicity for six endpoints (bovine serum albumin, carbonic anhydrase, chymotrypsin, hemoglobin along with cell viability and nitrogen oxide production) have been extracted from the corresponding optimized QSAR models. The molecular features relevant to each of the endpoint respective mechanism of action for the decorated nanotubes are also discussed. Based on the molecular information contained within the optimal QSAR models for each nanotoxicity endpoint, either the decorator attached to the nanotube is directly responsible for the expression of a particular activity, irrespective of the decorator's 3D-geometry and independent of the nanotube, or those decorators having structures that place the functional groups of the decorators as far as possible from the nanotube surface most strongly influence the biological activity. These molecular descriptors are further used to hypothesize specific interactions involved in the expression of each of the six biological endpoints. Copyright © 2015 Elsevier Inc. All rights reserved.

Spider silk MASP1 and MASP2 proteins as carbon fiber precursors

Energy Technology Data Exchange (ETDEWEB)

Lewis, Randolph V [Utah State Univ., Logan, UT (United States)

2017-06-14

. Task 2. Carbonization: The major accomplishment in the latter part of the work is that the ONRL group has successfully heated the spider silk protein fibers all the way up to 1700°C and produced a very competitive carbon fiber based on mechanical properties. Several important factors were discovered during these initial trials: 1) the ramp speed for increasing the temperature is critical; 2) maintaining tension on the fiber during the heating process because as it is heated it tends to expand; and 3) narrow temperature window in which stretching the fiber during heating leads to much better final materials. Task 3. Techno-Economic Methods: The techno-economic analysis was expanded to determine the relative cost of production with the bacterial production system compared to the transgenic alfalfa and goat production systems. The comparisons show two important things. For all systems the key factor in the final price is the amount of spider silk protein produced for whatever measure of volume or weight is used. Second alfalfa can be the cheapest but is subject to the possible regulatory control unless the US develops a more comprehensive approach to GMOs. The silkworm analysis was not completed due to a variety of confounding factors. The primary one was that if the production were shifted overseas then the cost would likely be nearly equivalent to current silk prices of $5-15/kg. However if concerns about the location of production is important then it would need to be done in the US and initial costs would be much higher but if the later scenario is utilized then the cost would be lowered but it was not possible to calculate exact costs.

The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

Directory of Open Access Journals (Sweden)

Bani Kumar Pathak

Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

Science.gov (United States)

Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

2010-09-15

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Fractionation of whey protein isolate with supercritical carbon dioxide – process modeling and cost estimation

Science.gov (United States)

An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of alpha-lactalbumin (alpha-La) and beta-lactoglobulin (beta-Lg) from a commercial whey protein isolate (WPI) containing 55% ...

A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

Energy Technology Data Exchange (ETDEWEB)

Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila (IP-Korea); (UPMC); (UMKC)

2013-09-23

The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

[Decomposition model of energy-related carbon emissions in tertiary industry for China].

Science.gov (United States)

Lu, Yuan-Qing; Shi, Jun

2012-07-01

Tertiary industry has been developed in recent years. And it is very important to find the factors influenced the energy-related carbon emissions in tertiary industry. A decomposition model of energy-related carbon emissions for China is set up by adopting logarithmic mean weight Divisia method based on the identity of carbon emissions. The model is adopted to analyze the influence of energy structure, energy efficiency, tertiary industry structure and economic output to energy-related carbon emissions in China from 2000 to 2009. Results show that the contribution rate of economic output and energy structure to energy-related carbon emissions increases year by year. Either is the contribution rate of energy efficiency or the tertiary industry restraining to energy-related carbon emissions. However, the restrain effect is weakening.

Interactions between proteins and carbon-based nanoparticles: exploring the origin of nanotoxicity at the molecular level.

Science.gov (United States)

Zuo, Guanghong; Kang, Seung-Gu; Xiu, Peng; Zhao, Yuliang; Zhou, Ruhong

2013-05-27

The widespread application of nanomaterials has spurred an interest in the study of interactions between nanoparticles and proteins due to the biosafety concerns of these nanomaterials. In this review, a summary is presented of some of the recent studies on this important subject, especially on the interactions of proteins with carbon nanotubes (CNTs) and metallofullerenols. Two potential molecular mechanisms have been proposed for CNTs' inhibition of protein functions. The driving forces of CNTs' adsorption onto proteins are found to be mainly hydrophobic interactions and the so-called π-π stacking between CNTs' carbon rings and proteins' aromatic residues. However, there is also recent evidence showing that endohedral metallofullerenol Gd@C82 (OH)22 can be used to inhibit tumor growth, thus acting as a potential nanomedicine. These recent findings have provided a better understanding of nanotoxicity at the molecular level and also suggested therapeutic potential by using nanoparticles' cytotoxicity against cancer cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Micromotor-Based Biomimetic Carbon Dioxide Sequestration: Towards Mobile Microscrubbers.

Science.gov (United States)

Uygun, Murat; Singh, Virendra V; Kaufmann, Kevin; Uygun, Deniz A; de Oliveira, Severina D S; Wang, Joseph

2015-10-26

We describe a mobile CO2 scrubbing platform that offers a greatly accelerated biomimetic sequestration based on a self-propelled carbonic anhydrase (CA) functionalized micromotor. The CO2 hydration capability of CA is coupled with the rapid movement of catalytic micromotors, and along with the corresponding fluid dynamics, results in a highly efficient mobile CO2 scrubbing microsystem. The continuous movement of CA and enhanced mass transport of the CO2 substrate lead to significant improvements in the sequestration efficiency and speed over stationary immobilized or free CA platforms. This system is a promising approach to rapid and enhanced CO2 sequestration platforms for addressing growing concerns over the buildup of greenhouse gas. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of ocean acidification on the physiological performance and carbon production of the Antarctic sea ice diatom Nitzschia sp. ICE-H.

Science.gov (United States)

Qu, Chang-Feng; Liu, Fang-Ming; Zheng, Zhou; Wang, Yi-Bin; Li, Xue-Gang; Yuan, Hua-Mao; Li, Ning; An, Mei-Ling; Wang, Xi-Xi; He, Ying-Ying; Li, Lu-Lu; Miao, Jin-Lai

2017-07-15

Ocean acidification (OA) resulting from increasing atmospheric CO 2 strongly influences marine ecosystems, particularly in the polar ocean due to greater CO 2 solubility. Here, we grew the Antarctic sea ice diatom Nitzschia sp. ICE-H in a semicontinuous culture under low (~400ppm) and high (1000ppm) CO 2 levels. Elevated CO 2 resulted in a stimulated physiological response including increased growth rates, chlorophyll a contents, and nitrogen and phosphorus uptake rates. Furthermore, high CO 2 enhanced cellular particulate organic carbon production rates, indicating a greater shift from inorganic to organic carbon. However, the cultures grown in high CO 2 conditions exhibited a decrease in both extracellular and intracellular carbonic anhydrase activity, suggesting that the carbon concentrating mechanisms of Nitzschia sp. ICE-H may be suppressed by elevated CO 2 . Our results revealed that OA would be beneficial to the survival of this sea ice diatom strain, with broad implications for global carbon cycles in the future ocean. Copyright © 2017. Published by Elsevier Ltd.

Mechanical desorption of immobilized proteins using carbon dioxide aerosols for reusable biosensors

Energy Technology Data Exchange (ETDEWEB)

Singh, Renu; Hong, Seongkyeol [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Jang, Jaesung, E-mail: jjang@unist.ac.kr [School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of); School of Materials Science and Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798 (Korea, Republic of)

2015-01-01

Highlights: • Immobilized proteins were removed using carbon dioxide aerosols. • We observed high removal efficiencies due to the aerosol treatment. • We confirmed the removal with FTIR and X-ray photoelectron spectroscopy. • This CO{sub 2} aerosol treatment did not undermine re-functionalization. • This technique is a fast and damage-free method to reuse a sensor surface. - Abstract: Reusability of a biosensor has recently received considerable attention, and it is closely related with the effective desorption of probe molecules. We present a novel mechanical desorption technique to reuse biosensors by using periodic jets of carbon dioxide (CO{sub 2}) aerosols (a mixture of solid and gaseous CO{sub 2}), and demonstrate its feasibility by removing physically adsorbed and covalently bonded fluorescent proteins i.e., Escherichia coli fluorescein isothiocyanate antibody and bovine serum albumin (E. coli FITC–Ab and FITC–BSA) from silicon chips. The proteins on the chip surfaces were measured by fluorescent images before and after applying the aerosols. The removal efficiency of the aerosol treatment was measured for various concentrations (1–20 μg mL{sup −1}) of E. coli FITC–Ab and FITC–BSA with two different removal cycles (5 and 11 cycles; each cycle: 8 s). We observed high removal efficiencies (>93.5% for physically adsorbed Ab and >84.6% for covalently bonded Ab) at 11 cycle aerosol treatment. This CO{sub 2} aerosol treatment did not undermine re-functionalization, which was confirmed by the fluorescent images of FITC–Abs for fresh and reused chips. Desorption of the immobilized layers was validated by Fourier transform infrared and X-ray photoelectron spectroscopic analyses. We also conducted an experiment on the regeneration of E. coli sensing chips using this aerosol treatment, and the chips were re-used 5 times successfully. This mechanical desorption technique is a highly effective and novel strategy for reusable biosensors.

Effects of CO2-driven acidification of seawater on the calcification process in the calcareous hydrozoan Millepora alcicornis (Linnaeus, 1758)

Science.gov (United States)

de Barros Marangoni, Laura Fernandes; Calderon, Emiliano Nicolas; Marques, Joseane Aparecida; Duarte, Gustavo Adolpho Santos; Pereira, Cristiano Macedo; e Castro, Clovis Barreira; Bianchini, Adalto

2017-12-01

Ocean acidification is expected to intensify due to increasing levels in the partial pressure of atmospheric CO2 ( pCO2). This could negatively affect major calcifying reef organisms. In this study, the effects of different levels of CO2-driven acidification of seawater (control: pH 8.1; moderate: pH 7.8; intermediate: pH 7.5; and severe: pH 7.2) on the net calcification rate and activity of enzymes related to the calcification process (Ca-ATPase and carbonic anhydrase) were evaluated in the calcareous hydrozoan Millepora alcicornis. The experiment was run for 30 d using a marine mesocosm system. Net calcification ratio was significantly reduced in hydrocorals exposed to intermediate seawater acidification for 16 d and to severe seawater acidification for 16 d or 30 d, compared to animals at control conditions. However, only hydrocorals exposed to severe seawater acidification showed lower net calcification rates than those exposed to control conditions for 30 d. In accordance, the activities of enzymes involved in the calcification process markedly increased in hydrocorals exposed to reduced pH. Ca-ATPase seemed to be more sensitive to seawater acidification than carbonic anhydrase as it increased in hydrocorals exposed to intermediate and severe seawater acidification for 30 d, while carbonic anhydrase activity was only stimulated under severe seawater acidification. Therefore, our findings clearly show that the hydrocoral M. alcicornis is able to cope, to some extent, with long-term CO2-driven acidification of seawater (pH ≥ 7.5). In addition, they show that Ca-ATPase plays a key role in the maintenance of calcification rate under scenarios of moderate and intermediate levels of seawater acidification. However, the observed increase in Ca-ATPase and carbonic anhydrase activity was not enough to compensate for the effects of CO2-driven reduction in seawater pH on the net calcification rate of the hydrocoral M. alcicornis under a scenario of severe ocean

Proteomic identification of differentially expressed proteins during alfalfa (Medicago sativa L. flower development

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Lingling Chen

2016-10-01

Full Text Available Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L. seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1, pollination (S2, and the post-pollination senescence period (S3. Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD. Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs, carbonic anhydrase (CA, and NADPH: quinone oxidoreductase-like protein (NQOLs. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials.

Science.gov (United States)

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

2013-12-01

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil-cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.

Muscle damage induced by stretch-shortening cycle exercise.

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Kyröläinen, H; Takala, T E; Komi, P V

1998-03-01

Strenuous stretch-shortening cycle exercise was used as a model to study the leakage of proteins from skeletal muscle. The analysis included serum levels of creatine kinase (S-CK), myoglobin (S-Mb), and carbonic anhydrase (S-CA III). Blood samples from power- (N=11) and endurance-trained (N=10) athletes were collected before, 0, and 2 h after the exercise, which consisted of a total of 400 jumps. The levels of all determined myocellular proteins increased immediately after the exercise (P exercise, and the ratio of S-CA III and S-Mb decreased (P recruitment order of motor units, and/or differences in training background.

Biocompatibility of Soft-Templated Mesoporous Carbons

Energy Technology Data Exchange (ETDEWEB)

Gencoglu, Maria F. [Michigan Technological Univ., Houghton, MI (United States). Dept. of Chemical Engineering; Spurri, Amanda [Widener Univ., Chester, PA (United States). Dept. of Chemical Engineering; Franko, Mitchell [Widener Univ., Chester, PA (United States). Dept. of Chemical Engineering; Chen, Jihua [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Hensley, Dale K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Nanophase Materials Science (CNMS); Heldt, Caryn L. [Michigan Technological Univ., Houghton, MI (United States). Dept. of Chemical Engineering; Saha, Dipendu [Widener Univ., Chester, PA (United States). Dept. of Chemical Engineering

2014-08-21

We report that soft-templated mesoporous carbon is morphologically a non-nano type of carbon. It is a relatively newer variety of biomaterial, which has already demonstrated its successful role in drug delivery applications. To investigate the toxicity and biocompatibility, we introduced three types of mesoporous carbons with varying synthesis conditions and pore textural properties. We compared the Brunauer–Emmett–Teller (BET) surface area and pore width and performed cytotoxicity experiments with HeLa cells, cell viability studies with fibroblast cells and hemocomapatibility studies. Cytotoxicity tests reveal that two of the carbons are not cytotoxic, with cell survival over 90%. The mesoporous carbon with the highest surface area showed slight toxicity (~70% cell survival) at the highest carbon concentration of 500 μg/mL. Fibroblast cell viability assays suggested high and constant viability of over 98% after 3 days with no apparent relation with materials property and good visible cell-carbon compatibility. No hemolysis (<1%) was confirmed for all the carbon materials. Protein adsorption experiments with bovine serum albumin (BSA) and fibrinogen revealed a lower protein binding capacity of 0.2–0.6 mg/m² and 2–4 mg/m² for BSA and fibrinogen, respectively, with lower binding associated with an increase in surface area. The results of this study confirm the biocompatibility of soft-templated mesoporous carbons.

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

International Nuclear Information System (INIS)

Lin, Fengmin; Yu, Shiyong; Gu, Le; Zhu, Xuetao; Wang, Jianshe; Zhu, Han; Lu, Yi; Wang, Yihua; Deng, Yulin; Geng, Lina

2015-01-01

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity. (author)

The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

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La Rosa, Ruggero; de la Peña, Fernando; Prieto, María Axiliadora; Rojo, Fernando

2014-01-01

Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Silk Spinning in Silkworms and Spiders.

Science.gov (United States)

Andersson, Marlene; Johansson, Jan; Rising, Anna

2016-08-09

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.

Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Patel, TN; Park, AHA; Bantat, S

2014-12-01

The limited permeability of the E. coli outer membrane can significantly hinder whole-cell biocatalyst performance. In this study, the SARS coronavirus small envelope protein (SCVE) was expressed in E. coli cells previously engineered for periplasmic expression of carbonic anhydrase (CA) activity. This maneuver increased small molecule uptake by the cells, resulting in increased apparent CA activity of the biocatalysts. The enhancements in activity were quantified using methods developed for traditional heterogeneous catalysis. The expression of the SCVE protein was found to significantly reduce the Thiele moduli (phi), as well as increase the effectiveness factors (eta), effective diffusivities (D-e), and permeabilities (P) of the biocatalysts. These catalytic improvements translated into superior performance of the biocatalysts for the precipitation of calcium carbonate from solution which is an attractive strategy for long-term sequestration of captured carbon dioxide. Overall, these results demonstrate that synthetic biology approaches can be used to enhance heterogeneous catalysts incorporated into microbial whole-cell scaffolds.

A time-series analysis of energy-related carbon emissions in Korea

International Nuclear Information System (INIS)

Ki-Hong Choi; Ang, B.W.

2001-01-01

Energy-related carbon emissions and their relationships with energy consumption and GNP in Korea are studied from 1961 to 1998. The ratio of carbon emissions to GNP is expressed as the product of the aggregate carbon factor and the energy intensity. Changes in the aggregate carbon factor are decomposed into the impacts associated with the fuel carbon factor and the fuel mix, using the Divisia index approach. The analysis is carried out using two sets of data, with and without wood consumption as an energy source, and very different results are obtained. This shows that carbon emission studies for developing countries based on commercial energy consumption only may have to be interpreted with caution. Our analysis also reveals that the impact of the energy intensity on carbon emissions is greater than that of the aggregate carbon factor. This finding supports the assertion made in earlier studies that the energy intensity is a more meaningful indicator than the aggregate carbon factor in the study of climate change resulting from energy-related emissions. (author)

Synthesis, Properties, and Applications of Low-Dimensional Carbon-Related Nanomaterials

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Ali Mostofizadeh

2011-01-01

Full Text Available In recent years, many theoretical and experimental studies have been carried out to develop one of the most interesting aspects of the science and nanotechnology which is called carbon-related nanomaterials. The goal of this paper is to provide a review of some of the most exciting and important developments in the synthesis, properties, and applications of low-dimensional carbon nanomaterials. Carbon nanomaterials are formed in various structural features using several different processing methods. The synthesis techniques used to produce specific kinds of low-dimensional carbon nanomaterials such as zero-dimensional carbon nanomaterials (including fullerene, carbon-encapsulated metal nanoparticles, nanodiamond, and onion-like carbons, one-dimensional carbon nanomaterials (including carbon nanofibers and carbon nanotubes, and two-dimensional carbon nanomaterials (including graphene and carbon nanowalls are discussed in this paper. Subsequently, the paper deals with an overview of the properties of the mainly important products as well as some important applications and the future outlooks of these advanced nanomaterials.

Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

Science.gov (United States)

Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

2017-08-01

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Carbonation-Related Microstructural Changesin Long-Term Durability Concrete

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Cláudio A. Rigo da Silva

2002-09-01

Full Text Available This paper discusses the effects of carbonation on the microstructure of Portland cement concrete for long-term durability applications. A class C40 concrete (characteristic compression strength between 40 MPa and 44 MPa on the 28th day, according to Brazilian standard NBR 8953 was chosen for the experimental study of the carbonation effects, from which test samples were molded for accelerated test under a 100%-CO2 atmosphere after physical and mechanical characterization. It was observed that carbonation provoked a reduction of 5% to 12% of the concrete open porosity accessible to water. Flexural strength values obtained after the carbonation tests revealed a decrease of 12% and 25% in relation to the values obtained before tests on the 28th and 91st days, respectively.

Cytoplasmic Histidine Kinase (HP0244)-Regulated Assembly of Urease with UreI, a Channel for Urea and Its Metabolites, CO2, NH3, and NH4+, Is Necessary for Acid Survival of Helicobacter pylori▿

OpenAIRE

Scott, David R.; Marcus, Elizabeth A.; Wen, Yi; Singh, Siddarth; Feng, Jing; Sachs, George

2009-01-01

Helicobacter pylori colonizes the normal human stomach by maintaining both periplasmic and cytoplasmic pH close to neutral in the presence of gastric acidity. Urease activity, urea flux through the pH-gated urea channel, UreI, and periplasmic α-carbonic anhydrase are essential for colonization. Exposure to pH 4.5 for up to 180 min activates total bacterial urease threefold. Within 30 min at pH 4.5, the urease structural subunits, UreA and UreB, and the Ni2+ insertion protein, UreE, are recrui...

Annotating activation/inhibition relationships to protein-protein interactions using gene ontology relations.

Science.gov (United States)

Yim, Soorin; Yu, Hasun; Jang, Dongjin; Lee, Doheon

2018-04-11

Signaling pathways can be reconstructed by identifying 'effect types' (i.e. activation/inhibition) of protein-protein interactions (PPIs). Effect types are composed of 'directions' (i.e. upstream/downstream) and 'signs' (i.e. positive/negative), thereby requiring directions as well as signs of PPIs to predict signaling events from PPI networks. Here, we propose a computational method for systemically annotating effect types to PPIs using relations between functional information of proteins. We used regulates, positively regulates, and negatively regulates relations in Gene Ontology (GO) to predict directions and signs of PPIs. These relations indicate both directions and signs between GO terms so that we can project directions and signs between relevant GO terms to PPIs. Independent test results showed that our method is effective for predicting both directions and signs of PPIs. Moreover, our method outperformed a previous GO-based method that did not consider the relations between GO terms. We annotated effect types to human PPIs and validated several highly confident effect types against literature. The annotated human PPIs are available in Additional file 2 to aid signaling pathway reconstruction and network biology research. We annotated effect types to PPIs by using regulates, positively regulates, and negatively regulates relations in GO. We demonstrated that those relations are effective for predicting not only signs, but also directions of PPIs. The usefulness of those relations suggests their potential applications to other types of interactions such as protein-DNA interactions.

Nanocarrier-mediated foliar zinc fertilization influences expression of metal homeostasis related genes in flag leaves and enhances gluten content in durum wheat.

Science.gov (United States)

Deshpande, Paresh; Dapkekar, Ashwin; Oak, Manoj; Paknikar, Kishore; Rajwade, Jyutika

2018-01-01

Wheat is the staple food for most of the world's population; however, it is a poor source of zinc. Foliar fertilization of zinc via zinc loaded chitosan nanocarriers (Zn-CNP) post-anthesis has proved to be a promising approach for grain zinc enhancement in durum wheat as evidenced in our earlier study. However, the molecular mechanism of uptake of zinc via Zn-CNP remains unclear. Foliar application of Zn-CNP was performed at post anthesis stages in two durum wheat cultivars (MACS 3125 and UC1114, containing the Gpc-B1 gene), and expression levels of several metal-related genes were analyzed during early senescence. Zn-CNP application indeed caused changes in gene expression as revealed by qPCR data on representative genes involved in metal homeostasis, phloem transporters, and leaf senescence. Furthermore, zinc-regulated transporters and iron (Fe)-regulated transporter-like protein (ZIP) family [ZIP1, ZIP7, ZIP15], CA (carbonic anhydrase), and DMAS (2'-deoxymugineic acid synthase) in flag leaves exhibited significant correlation with zinc content in the seeds. The analysis of grain endosperm proteins showed enhancement of gamma gliadins while other gluten subunits decreased. Gene expression within ZIP family members varied with the type of cultivar mostly attributed to the Gpc-B1, concentration of external zinc ions as well as the type of tissue analyzed. Correlation analysis revealed the involvement of the selected genes in zinc enhancement. At the molecular level, uptake of zinc via Zn-CNP nanocarrier was comparable to the uptake of zinc via common zinc fertilizers i.e. ZnSO4.

Nanocarrier-mediated foliar zinc fertilization influences expression of metal homeostasis related genes in flag leaves and enhances gluten content in durum wheat.

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Paresh Deshpande

Full Text Available Wheat is the staple food for most of the world's population; however, it is a poor source of zinc. Foliar fertilization of zinc via zinc loaded chitosan nanocarriers (Zn-CNP post-anthesis has proved to be a promising approach for grain zinc enhancement in durum wheat as evidenced in our earlier study. However, the molecular mechanism of uptake of zinc via Zn-CNP remains unclear.Foliar application of Zn-CNP was performed at post anthesis stages in two durum wheat cultivars (MACS 3125 and UC1114, containing the Gpc-B1 gene, and expression levels of several metal-related genes were analyzed during early senescence. Zn-CNP application indeed caused changes in gene expression as revealed by qPCR data on representative genes involved in metal homeostasis, phloem transporters, and leaf senescence. Furthermore, zinc-regulated transporters and iron (Fe-regulated transporter-like protein (ZIP family [ZIP1, ZIP7, ZIP15], CA (carbonic anhydrase, and DMAS (2'-deoxymugineic acid synthase in flag leaves exhibited significant correlation with zinc content in the seeds. The analysis of grain endosperm proteins showed enhancement of gamma gliadins while other gluten subunits decreased. Gene expression within ZIP family members varied with the type of cultivar mostly attributed to the Gpc-B1, concentration of external zinc ions as well as the type of tissue analyzed. Correlation analysis revealed the involvement of the selected genes in zinc enhancement.At the molecular level, uptake of zinc via Zn-CNP nanocarrier was comparable to the uptake of zinc via common zinc fertilizers i.e. ZnSO4.

Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

DEFF Research Database (Denmark)

Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian

2006-01-01

Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

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Shuaizheng Jia

Full Text Available The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

Brain carbonic acid acidosis after acetazolamide

DEFF Research Database (Denmark)

Heuser, D; Astrup, J; Lassen, N A

1975-01-01

acidosis by I.V. injection of HCO3-minus. Acetazolamide (25 mg/kg) i.v. was followed by a marked brain acidosis which after 10 min had progressed to a drop in pH of 0.203 plus or minus 0.046 (x bar plus or minus S.D., n equals 8). The slowness ofthe development of acidosis points to a direct effect......In cats in barbiturate anesthesia extracellular pH and potassium were continously recorded from brian cortex by implanted microelectrodes. Implantation of the electrodes preserved the low permeability of the blood-brain-barrier to HCO3-minus and H+ions as indicated by the development of brain...... of the carbonic anhydrase inhibition on the brain tissue. As a further support for this conclusion was considered the finding of a prolonged response time of brain pH to HCO3-minus i.v. to CO2-minus inhalation, and to hyperventilation after the acetazolamide inhibtion. No changes in brain extracelllular potassium...

Production and characterization of biodiesel from carbon dioxide concentrating chemolithotrophic bacteria, Serratia sp. ISTD04.

Science.gov (United States)

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

2014-02-01

A chemolithotrophic bacterium, Serratia sp. ISTD04, enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was evaluated for potential of carbon dioxide (CO2) sequestration and biofuel production. CO2 sequestration efficiency of the bacterium was determined by enzymatic activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further, Western blot analysis confirmed presence of RuBisCO. The bacterium produced 0.487 and 0.647mgmg(-1) per unit cell dry weight of hydrocarbons and lipids respectively. The hydrocarbons were within the range of C13-C24 making it equivalent to light oil. GC-MS analysis of lipids produced by the bacterium indicated presence of C15-C20 organic compounds that made it potential source of biodiesel after transesterification. GC-MS, FTIR and NMR spectroscopic characterization of the fatty acid methyl esters revealed the presence of 55% and 45% of unsaturated and saturated organic compounds respectively, thus making it a balanced biodiesel composition. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stromal Expression of Hypoxia Regulated Proteins Is an Adverse Prognostic Factor in Colorectal Carcinomas

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Arjen H. G. Cleven

2007-01-01

Full Text Available Background: Hypoxia modifies the phenotype of tumors in a way that promotes tumor aggressiveness and resistance towards chemotherapy and radiotherapy. However, the expression and influence of hypoxia-regulated proteins on tumor biology are not well characterized in colorectal tumors. We studied the role of protein expression of hypoxia-inducible factor (HIF-1α, HIF-2α, carbonic anhydrase 9 (CA9 and glucose transporter 1 (GLUT1 in patients with colorectal adenocarcinomas. Methods: Expression of HIF-1α, HIF-2α, CA9 and GLUT1 was quantified by immunohistochemistry in 133 colorectal adenocarcinomas. The expression of hypoxia markers was correlated with clinicopathological variables and overall patient survival. Results: Expression of these hypoxia markers was detected in the epithelial compartment of the tumor cells as well as in tumor-associated stromal cells. Although tumor cells frequently showed expression of one or more of the investigated hypoxia markers, no correlation among these markers or with clinical response was found. However, within the tumor stroma, positive correlations between the hypoxia markers HIF-2α, CA9 and GLUT1 were observed. Furthermore expression of HIF-2α and CA9 in tumor-associated stroma were both associated with a significantly reduced overall survival. In the Cox proportional hazard model, stromal HIF-2α expression was an independent prognostic factor for survival. Conclusion: These observations show, that expression of hypoxia regulated proteins in tumor-associated stromal cells, as opposed to their expression in epithelial tumor cells, is associated with poor outcome in colorectal cancer. This study suggests that tumor hypoxia may influence tumor-associated stromal cells in a way that ultimately contributes to patient prognosis.

Height-related growth declines in ponderosa pine are not due to carbon limitation.

Science.gov (United States)

Sala, Anna; Hoch, Günter

2009-01-01

Decreased gas exchange as trees grow tall has been proposed to explain age-related growth declines in trees. We examined changes of mobile carbon stores (starch, sugars and lipids) with tree height in ponderosa pine (Pinus ponderosa) at two sites differing in water availability, and tested the following hypotheses: (1) carbon supply does not become increasingly limited as trees grow tall; rather, the concentration of mobile carbon compounds increases with tree height reflecting greater reductions of carbon sink activities relative to carbon assimilation; and (2) increases of stored mobile carbon compounds with tree height are greater in drier sites. Height-related growth reductions were associated with significant increases of non-structural carbohydrates (NSC) and lipid concentrations in all tissues in the upper canopy and of NSC in the bole. Lipid concentrations in the bole decreased with tree height, but such decrease is not necessarily inconsistent with non-limiting carbon supply in tall trees. Furthermore, we found stronger increases of mobile carbon stores with tree height at the dry site relative to the moist site. Our results provide first direct evidence that carbon supply does not limit growth in tall trees and that decreases of water availability might negatively impact growth processes more than net-photosynthesis.

Actin, actin-binding proteins, and actin-related proteins in the nucleus.

Science.gov (United States)

Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

2016-04-01

Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

Energetics investigation on encapsulation of protein/peptide drugs in carbon nanotubes.

Science.gov (United States)

Chen, Qu; Wang, Qi; Liu, Ying-Chun; Wu, Tao; Kang, Yu; Moore, Joshua D; Gubbins, Keith E

2009-07-07

This work focuses on the dynamic properties and energetics of the protein/peptide drug during its transport through carbon nanotubes (CNTs). A systematic study was performed on the interaction between the peptide and the CNTs. In the molecular dynamics (MD) simulations, the protein/peptide molecule Zadaxin is observed to be encapsulated inside the nanotube after its spontaneous insertion and oscillates around the center of the tube, where the van der Waals interaction energy is observed to be a minimum. Furthermore, it is found by performing steered MD simulations that the pulling force applied to the peptide reaches a maximum value, which demonstrates the ability of the CNTs to trap protein/peptide drugs. Such effects, attributed to van der Waals interactions, can be influenced by varying the lengths and diameters of the CNTs. Longer nanotubes provide a broader area to trap the peptide, while smaller nanotubes are able to encapsulate the peptide with a deeper interaction energy well. This investigation provides insights into nanoscale pharmaceutical drug delivery devices.

Carbon diffusion behavior in molybdenum at relatively low temperatures

Energy Technology Data Exchange (ETDEWEB)

Hiraoka, Yutaka, E-mail: hiraoka@dap.ous.ac.j [Department of Applied Physics, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005 (Japan); Imamura, Kyosuke [Graduate School of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005 (Japan); Kadokura, Takanori; Yamamoto, Yoshiharu [Materials Research Department, A.L.M.T. Corp., 2 Iwasekoshi-machi, Toyama 931-8543 (Japan)

2010-01-07

Purpose of this study is to investigate the carbon diffusion behavior in pure molybdenum at relatively low temperatures by means of fracture surface observation. Carbon addition was performed at a temperature of 1273-1373 K with the heating time being changed. Fracture surface of the specimen after carbon addition was examined using SEM and the carbon diffusion distance was estimated from the change of fracture mode as a function of the distance from the surface. Results are summarized as follows. First, the carbon diffusion distance increased approximately linearly with the increase of heating time from 1.2 to 10.8 ks. This relationship does not agree with that obtained at much higher temperatures. From Arrhenius plots of the slope of the straight line and the temperature, activation energy was calculated (155 kJ/mol). Secondly, the carbon diffusion distance estimated in this study was generally larger than that simulated using the data of Rudman, particularly at a longer heating time.

Progress in 3D Printing of Carbon Materials for Energy-Related Applications.

Science.gov (United States)

Fu, Kun; Yao, Yonggang; Dai, Jiaqi; Hu, Liangbing

2017-03-01

The additive-manufacturing (AM) technique, known as three-dimensional (3D) printing, has attracted much attention in industry and academia in recent years. 3D printing has been developed for a variety of applications. Printable inks are the most important component for 3D printing, and are related to the materials, the printing method, and the structures of the final 3D-printed products. Carbon materials, due to their good chemical stability and versatile nanostructure, have been widely used in 3D printing for different applications. Good inks are mainly based on volatile solutions having carbon materials as fillers such as graphene oxide (GO), carbon nanotubes (CNT), carbon blacks, and solvent, as well as polymers and other additives. Studies of carbon materials in 3D printing, especially GO-based materials, have been extensively reported for energy-related applications. In these circumstances, understanding the very recent developments of 3D-printed carbon materials and their extended applications to address energy-related challenges and bring new concepts for material designs are becoming urgent and important. Here, recent developments in 3D printing of emerging devices for energy-related applications are reviewed, including energy-storage applications, electronic circuits, and thermal-energy applications at high temperature. To close, a conclusion and outlook are provided, pointing out future designs and developments of 3D-printing technology based on carbon materials for energy-related applications and beyond. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elevated temperature alters carbon cycling in a model microbial community

Science.gov (United States)

Mosier, A.; Li, Z.; Thomas, B. C.; Hettich, R. L.; Pan, C.; Banfield, J. F.

2013-12-01

Earth's climate is regulated by biogeochemical carbon exchanges between the land, oceans and atmosphere that are chiefly driven by microorganisms. Microbial communities are therefore indispensible to the study of carbon cycling and its impacts on the global climate system. In spite of the critical role of microbial communities in carbon cycling processes, microbial activity is currently minimally represented or altogether absent from most Earth System Models. Method development and hypothesis-driven experimentation on tractable model ecosystems of reduced complexity, as presented here, are essential for building molecularly resolved, benchmarked carbon-climate models. Here, we use chemoautotropic acid mine drainage biofilms as a model community to determine how elevated temperature, a key parameter of global climate change, regulates the flow of carbon through microbial-based ecosystems. This study represents the first community proteomics analysis using tandem mass tags (TMT), which enable accurate, precise, and reproducible quantification of proteins. We compare protein expression levels of biofilms growing over a narrow temperature range expected to occur with predicted climate changes. We show that elevated temperature leads to up-regulation of proteins involved in amino acid metabolism and protein modification, and down-regulation of proteins involved in growth and reproduction. Closely related bacterial genotypes differ in their response to temperature: Elevated temperature represses carbon fixation by two Leptospirillum genotypes, whereas carbon fixation is significantly up-regulated at higher temperature by a third closely related genotypic group. Leptospirillum group III bacteria are more susceptible to viral stress at elevated temperature, which may lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, this proteogenomics approach revealed the effects of climate change on carbon cycling pathways and other

Carbon nanotubes-assisted polyacrylamide gel electrophoresis for enhanced separation of human serum proteins and application in liverish diagnosis.

Science.gov (United States)

Jiang, Fubin; Wang, Yanan; Hu, Xinfang; Shao, Na; Na, Na; Delanghe, Joris R; Ouyang, Jin

2010-11-01

The application of pore-gradient polyacrylamide gel electrophoresis (PG-PAGE) incorporated with carbon nanotube modified by Triton X-100 and carboxylation so as to improve the separation of human serum proteins is reported. The novel PG-PAGE was made by adding water-soluble single-walled carbon nanotubes (CNTs) when preparing the polyacrylamide gel. Significant improvements in separation of complement C3 protein and haptoglobin (Hp) in human serum were achieved. It was estimated that the interactions between the hydrophilic groups on the proteins and the surface of the CNTs result in different adsorption kinetics of complement C3 and Hp subtype on the nanoparticles incorporated in the gel, thus enhancing the separation of the two proteins in serum. This new CNT matrix-assisted PG-PAGE method for enhanced separation of complement C3 and Hp in human serum was successfully applied to distinguish the samples from liverish patients and healthy people.

Functionalization of single-walled carbon nanotubes with protein by click chemistry as sensing platform for sensitized electrochemical immunoassay

International Nuclear Information System (INIS)

Qi Honglan; Ling Chen; Huang Ru; Qiu Xiaoying; Shangguan Li; Gao Qiang; Zhang Chengxiao

2012-01-01

Highlights: ► Single-walled carbon nanotubes were functionalized with protein by click chemistry. ► The SWNTs conjugated with protein showed excellent dispersion in water and kept good bioacitvity. ► A competitive electrochemical immunoassay for the determination of anti-IgG was developed with high sensitivity and good stability. - Abstract: The application of the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition to the functionalization of single-walled carbon nanotubes (SWNTs) with the protein and the use of the artificial SWNTs as a sensing platform for sensitive immunoassay were reported. Covalent functionalization of azide decorated SWNTs with alkyne modified protein was firstly accomplished by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron micrograph were used to characterize the protein-functionalized SWNTs. It was found that the SWNTs conjugated with the proteins showed excellent dispersion in water and kept good bioacitivity when immunoglobulin (IgG) and horseradish peroxidase (HRP) were chosen as model proteins. As a proof-of-concept, IgG-functionalized SWNTs were immobilized onto the surface of a glassy carbon electrode by simple casting method as immunosensing platform and a sensitive competitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using HRP as enzyme label. The fabrication of the immunosensor were characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the redox probe [Fe(CN) 6 ] 3−/4− . The SWNTs as immobilization platform showed better sensitizing effect, a detection limit of 30 pg mL −1 (S/N = 3) was obtained for anti-IgG. The proposed strategy provided a stable immobilization method and sensitized recognition platform for analytes. This work demonstrated that the click coupling of SWNTs with protein was an effective

The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

NARCIS (Netherlands)

Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

Photosynthetic carbon metabolism in freshwater phytoplankton

International Nuclear Information System (INIS)

Groeger, A.W.

1986-01-01

Photosynthetic carbon metabolism of natural assemblages of freshwater phytoplankton was measured by following the flow of inorganic 14 C into the photosynthetic end products polysaccharide protein, lipid, and soluble metabolites. Data were collected from a wide range of physical, chemical, and trophic conditions in six southern United States reservoirs, with the primary environmental variables of interest being light intensity and nutrient supply. Polysaccharide and protein were consistently the primary products of photosynthetic carbon metabolism, comprising an average of 70% of the total carbon fixation over a wide range of light intensities. Polysaccharide was quantitatively more important at higher light intensities, and protein at lower light intensities, as light intensity varied both with depth within the water column and over diurnal cycles. Polysaccharide synthesis was more variable over the diurnal period than was protein synthesis. Phytoplankton in the downlake epilimnion of Normandy Lake, a central Tennessee reservoir, responded to summer nitrogen (N) deficiency by increasing relative rates of lipid synthesis from 10-15% to 20-25% of the total photosynthetic carbon fixation. Phytoplankton in more nitrogen-sufficient areas of the reservoir maintained lower rates of lipid synthesis throughout the summer. These results document the occurrence in nature of a relationship between N-deficiency and increased lipid synthesis previously observed only in laboratory algal culture studies

Protein arrangement on modified diamond-like carbon surfaces - An ARXPS study

Science.gov (United States)

Oosterbeek, Reece N.; Seal, Christopher K.; Hyland, Margaret M.

2014-12-01

Understanding the nature of the interface between a biomaterial implant and the biological fluid is an essential step towards creating improved implant materials. This study examined a diamond-like carbon coating biomaterial, the surface energy of which was modified by Ar+ ion sputtering and laser graphitisation. The arrangement of proteins was analysed by angle resolved X-ray photoelectron spectroscopy, and the effects of the polar component of surface energy on this arrangement were observed. It was seen that polar groups (such as CN, CO) are more attracted to the coating surface due to the stronger polar interactions. This results in a segregation of these groups to the DLC-protein interface; at increasing takeoff angle (further from to DLC-protein interface) fewer of these polar groups are seen. Correspondingly, groups that interact mainly by dispersive forces (CC, CH) were found to increase in intensity as takeoff angle increased, indicating they are segregated away from the DLC-protein interface. The magnitude of the segregation was seen to increase with increasing polar surface energy, this was attributed to an increased net attraction between the solid surface and polar groups at higher polar surface energy (γSp).

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

Genome-wide identification of regulatory elements and reconstruction of gene regulatory networks of the green alga Chlamydomonas reinhardtii under carbon deprivation.

Directory of Open Access Journals (Sweden)

Flavia Vischi Winck

Full Text Available The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1 gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF and transcription regulator (TR genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1 and Lcr2 (Low-CO2 response regulator 2, may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome

A protein relational database and protein family knowledge bases to facilitate structure-based design analyses.

Science.gov (United States)

Mobilio, Dominick; Walker, Gary; Brooijmans, Natasja; Nilakantan, Ramaswamy; Denny, R Aldrin; Dejoannis, Jason; Feyfant, Eric; Kowticwar, Rupesh K; Mankala, Jyoti; Palli, Satish; Punyamantula, Sairam; Tatipally, Maneesh; John, Reji K; Humblet, Christine

2010-08-01

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

Optic nerve pH and PO2

DEFF Research Database (Denmark)

Pedersen, Daniella B; Stefánsson, Einar; Kiilgaard, Jens Folke

2006-01-01

Earlier studies have demonstrated that carbonic anhydrase inhibitors (CAIs) increase optic nerve oxygen tension (ONPO(2)) in pigs. We hypothesized that the mechanism of this effect was either a CO(2) increase or a pH decrease in tissue and blood. To test this hypothesis we investigated and compared...... how optic nerve pH (ONpH) and ONPO(2) are affected by: (1) carbonic anhydrase inhibition; (2) respiratory acidosis, and (3) metabolic acidosis. We measured ONpH with a glass pH electrode and ONPO(2) with a polarographic oxygen electrode. One of the electrodes was placed in the vitreous cavity 0.5 mm...

Retinitis pigmentosa sine pigmenti. Debut with macular oedema.

Science.gov (United States)

de la Mata Pérez, G; Ruiz-Moreno, O; Fernández-Pérez, S; Torrón Fernández-Blanco, C; Pablo-Júlvez, L

2014-09-01

A 25-year-old woman, with metamorphopsia in her left eye of one year onset. The examination revealed a bilateral cystoid macular oedema (CME) and vascular attenuation. We describe the diagnostic tests, as well as differential diagnosis and treatment response with carbonic anhydrase inhibitors. The retinitis pigmentosa sine pigment is a subtype of atypical retinitis pigmentosa characterised by the absence of pigment deposits. The night blindness is milder, and perimetric and electroretinographic impairment is lower. CME is an important cause of central vision loss, and responds to anhydrase carbonic inhibitors. Copyright © 2012 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Relations between protein production, protein quality and environmental factors in Pisum mutants

International Nuclear Information System (INIS)

Gottschalk, W.; Mueller, H.P.; Wolff, G.

1975-01-01

The seed protein content of 138 radiation-induced Pisum mutants was determined. The variability of this genetically well-defined material agrees approximately with that of the world collection of Pisum sativum. Some environmental factors to a great extent influence the protein production of the mutants and the initial line. Therefore, it is necessary to consider the relations between the genetically controlled protein production and its dependence upon the environmental factors. This is especially evident if the protein situation of the same genotypes cultivated under the moderate climatic conditions of middle Europe is compared with the subtropical conditions of India. A generally firm correlation between seed size and protein content could not be found in material regarding 148 different mutants of our assortment. Therefore, the selection of small-grained mutants does not result in a selection of protein-rich genotypes in Pisum sativum. Considering all the criteria positively and negatively influencing the protein production, a positive situation could be found in some mutants, especially in the fasciated ones. Furthermore, an improvement of the protein quality could be reached by a genetically conditioned alteration of the globulin-albumin ratio leading to an increase of some essential amino acids such as methionine and lysine. The combined action of mutant genes results in unexpected changes of the protein quantity as well as the quality of the recombinants in relation to their parental mutants. The comparison of some essential amino acids of our useful mutants with those of the varieties of other genera of the Leguminosae shows certain trends of biochemical alterations realized during evolutionary development of the family. (author)

Synthesis, Properties, and Applications of Low-Dimensional Carbon-Related Nano materials

International Nuclear Information System (INIS)

Mostofizadeh, A.; Li, Y.; Song, B.; Huang, Y.; Mostofizadeh, A.

2011-01-01

In recent years, many theoretical and experimental studies have been carried out to develop one of the most interesting aspects of the science and nano technology which is called carbon-related nano materials. The goal of this paper is to provide a review of some of the most exciting and important developments in the synthesis, properties, and applications of low-dimensional carbon nano materials. Carbon nano materials are formed in various structural features using several different processing methods. The synthesis techniques used to produce specific kinds of low-dimensional carbon nano materials such as zero-dimensional carbon nano materials (including fullerene, carbon-encapsulated metal nanoparticles, nano diamond, and onion-like carbons), one-dimensional carbon nano materials (including carbon nano fibers and carbon nano tubes), and two-dimensional carbon nano materials (including graphene and carbon nano walls) are discussed in this paper. Subsequently, the paper deals with an overview of the properties of the mainly important products as well as some important applications and the future outlooks of these advanced nano materials.

Protein Biosensors Based on Polymer Nanowires, Carbon Nanotubes and Zinc Oxide Nanorods

Directory of Open Access Journals (Sweden)

Taeksoo Ji

2011-05-01

Full Text Available The development of biosensors using electrochemical methods is a promising application in the field of biotechnology. High sensitivity sensors for the bio-detection of proteins have been developed using several kinds of nanomaterials. The performance of the sensors depends on the type of nanostructures with which the biomaterials interact. One dimensional (1-D structures such as nanowires, nanotubes and nanorods are proven to have high potential for bio-applications. In this paper we review these three different kinds of nanostructures that have attracted much attention at recent times with their great performance as biosensors. Materials such as polymers, carbon and zinc oxide have been widely used for the fabrication of nanostructures because of their enhanced performance in terms of sensitivity, biocompatibility, and ease of preparation. Thus we consider polymer nanowires, carbon nanotubes and zinc oxide nanorods for discussion in this paper. We consider three stages in the development of biosensors: (a fabrication of biomaterials into nanostructures, (b alignment of the nanostructures and (c immobilization of proteins. Two different methods by which the biosensors can be developed at each stage for all the three nanostructures are examined. Finally, we conclude by mentioning some of the major challenges faced by many researchers who seek to fabricate biosensors for real time applications.

Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1α in human glioma in vitro and in vivo

International Nuclear Information System (INIS)

Said, Harun M.; Hagemann, Carsten; Staab, Adrian; Stojic, Jelena; Kuehnel, Siglinde; Vince, Giles H.; Flentje, Michael; Roosen, Klaus; Vordermark, Dirk

2007-01-01

Background and purpose: To identify molecular markers of tumor hypoxia and potential therapeutic targets in glioblastoma (GBM), we investigated the hypoxia-related expression of osteopontin (OPN), carbonic anhydrase 9 (CA9), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in vitro in human GBM cell lines and in vivo in human tumor samples of GBM, compared to low-grade astrocytoma (LGA). Materials and methods: Expression of the hypoxia-induced genes OPN, CA9, EPO, VEGF and HIF-1α was analyzed in three GBM cell lines, GaMG, U373 and U251, under in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O 2 ) and in tumor samples from two patient groups with LGA and GBM (n = 15 each), at the mRNA level (semiquantitative RT-PCR). Selected conditions and representative tumor samples were also evaluated at the protein level by Western blot. Results: OPN and CA9 mRNA was most consistently upregulated in relation to severity and duration of in vitro hypoxia. In tumor samples, mean expression levels (LGA vs. GBM, normalized to mean expression in normal brain) were 1.71 vs. 4.57 (p < 0.001) for OPN, 1.11 vs. 3.35 (p < 0.001) for CA9, 2.79 vs. 5.28 (not significant, n.s.) for Epo, 1.13 vs. 2.0 (p = 0.007) for VEGF and 0.97 vs. 0.97 (n.s.) for HIF-1α. In tumor samples, GBM showed a particularly strong protein expression of OPN. Conclusions: Among a panel of known hypoxia-inducible genes, OPN and CA9 emerge as most consistently induced by in vitro hypoxia in human GBM cell lines and most specifically expressed in patient GBM tumor tissue, rendering these two genes attractive targets for hypoxia-directed treatment approaches

Acute inhalation toxicity of carbonyl sulfide

Energy Technology Data Exchange (ETDEWEB)

Benson, J.M.; Hahn, F.F.; Barr, E.B. [and others

1995-12-01

Carbonyl sulfide (COS), a colorless gas, is a side product of industrial procedures sure as coal hydrogenation and gasification. It is structurally related to and is a metabolite of carbon disulfide. COS is metabolized in the body by carbonic anhydrase to hydrogen sulfide (H{sub 2}S), which is thought to be responsible for COS toxicity. No threshold limit value for COS has been established. Results of these studies indicate COS (with an LC{sub 50} of 590 ppm) is slightly less acutely toxic than H{sub 2}S (LC{sub 50} of 440 ppm).

Enhance the performance of current scoring functions with the aid of 3D protein-ligand interaction fingerprints.

Science.gov (United States)

Liu, Jie; Su, Minyi; Liu, Zhihai; Li, Jie; Li, Yan; Wang, Renxiao

2017-07-18

In structure-based drug design, binding affinity prediction remains as a challenging goal for current scoring functions. Development of target-biased scoring functions provides a new possibility for tackling this problem, but this approach is also associated with certain technical difficulties. We previously reported the Knowledge-Guided Scoring (KGS) method as an alternative approach (BMC Bioinformatics, 2010, 11, 193-208). The key idea is to compute the binding affinity of a given protein-ligand complex based on the known binding data of an appropriate reference complex, so the error in binding affinity prediction can be reduced effectively. In this study, we have developed an upgraded version, i.e. KGS2, by employing 3D protein-ligand interaction fingerprints in reference selection. KGS2 was evaluated in combination with four scoring functions (X-Score, ChemPLP, ASP, and GoldScore) on five drug targets (HIV-1 protease, carbonic anhydrase 2, beta-secretase 1, beta-trypsin, and checkpoint kinase 1). In the in situ scoring test, considerable improvements were observed in most cases after application of KGS2. Besides, the performance of KGS2 was always better than KGS in all cases. In the more challenging molecular docking test, application of KGS2 also led to improved structure-activity relationship in some cases. KGS2 can be applied as a convenient "add-on" to current scoring functions without the need to re-engineer them, and its application is not limited to certain target proteins as customized scoring functions. As an interpolation method, its accuracy in principle can be improved further with the increasing knowledge of protein-ligand complex structures and binding affinity data. We expect that KGS2 will become a practical tool for enhancing the performance of current scoring functions in binding affinity prediction. The KGS2 software is available upon contacting the authors.

The Carbonation of Wollastonite: A Model Reaction to Test Natural and Biomimetic Catalysts for Enhanced CO2 Sequestration

Directory of Open Access Journals (Sweden)

Fulvio Di Lorenzo

2018-05-01

Full Text Available One of the most promising strategies for the safe and permanent disposal of anthropogenic CO2 is its conversion into carbonate minerals via the carbonation of calcium and magnesium silicates. However, the mechanism of such a reaction is not well constrained, and its slow kinetics is a handicap for the implementation of silicate mineral carbonation as an effective method for CO2 capture and storage (CCS. Here, we studied the different steps of wollastonite (CaSiO3 carbonation (silicate dissolution → carbonate precipitation as a model CCS system for the screening of natural and biomimetic catalysts for this reaction. Tested catalysts included carbonic anhydrase (CA, a natural enzyme that catalyzes the reversible hydration of CO2(aq, and biomimetic metal-organic frameworks (MOFs. Our results show that dissolution is the rate-limiting step for wollastonite carbonation. The overall reaction progresses anisotropically along different [hkl] directions via a pseudomorphic interface-coupled dissolution–precipitation mechanism, leading to partial passivation via secondary surface precipitation of amorphous silica and calcite, which in both cases is anisotropic (i.e., (hkl-specific. CA accelerates the final carbonate precipitation step but hinders the overall carbonation of wollastonite. Remarkably, one of the tested Zr-based MOFs accelerates the dissolution of the silicate. The use of MOFs for enhanced silicate dissolution alone or in combination with other natural or biomimetic catalysts for accelerated carbonation could represent a potentially effective strategy for enhanced mineral CCS.

Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched alpha-lactalbumin and beta-lactoglobulin food ingredients

Science.gov (United States)

A potentially economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (SCO2) as an acid to produce enriched fractions of alpha-lactalbumin (a-LA) and beta-lactoglobulin (b-LG) from whey protein isolate. To prepare the fractions, so...

Syntheses of carbon-13 labeled protoporphyrin-IX for spectroscopic studies of heme proteins

International Nuclear Information System (INIS)

Fujinari, E.M.

1985-01-01

The development of various methodologies for synthesis of selectively tailored protoporphyrin-IX dimethyl ester are presented. The iron(II) complex of protoporphyrin-IX is the heme, the prosthetic group for Hb, Mb, cytochromes and peroxidases. The significance of this research is to provide direct means to establish definitive carbon-13 NMR assignments of heme proteins in order to study not only the structure-function relationships, but also protein dynamics of these vital systems. Carbon-13 labeling at the beta-vinyl position was first achieved by ozonolysis of protoporphyrin-IX dimethyl ester. Column LC method were used to first isolate 2,4-diformyldeuteroporphyrin-IX dimethyl ester. Concomitantly, monofomyl-monovinyl porphyrins were obtained as a mixture of two isomers. This mixture was separated by MPLC or prep HPLC to afford the isomerically pure products, Spirographis porphyrin dimethyl ester and Iso-Spirographis porphyrin dimethyl ester. A Wittig reaction to each of these porphyrins with 13 C-methyltriphenylphosphonium iodide gave 2,4-bis[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, 2-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, and the 4-[ 13 C 2 ]-vinyl protoporphyrin-IX dimethyl ester, respectively

Carbon Dioxide (CO2 Sequestration In Bio-Concrete, An Overview

Directory of Open Access Journals (Sweden)

Faisal Alshalif A.

2017-01-01

Full Text Available The emission of CO2 into atmosphere which has increased rapidly in the last years has led to global warming. Therefore, in order to overcome the negative impacts on human and environment, the researchers focused mainly on the reduction and stabilization of CO2 which represent the main contributor in the increasing global warming. The natural capturing and conversion of CO2 from atmosphere is taken place by biological, chemical and physical processes. However, these processes need long time to cause a significant reduction in CO2. Recently, scientists shifted to use green technologies that aimed to produce concrete with high potential to adsorb CO2 in order to accelerate the reduction of CO2. In the present review the potential of bio-concrete to sequestrate CO2 based on carbonation process and as a function of carbonic anhydrase (CA is highlighted. The factors affecting CO2 sequestration in concrete and bacterial species are discussed. It is evident from the literatures, that the new trends to use bio-concrete might contribute in the reduction of CO2 and enhance the strength of non-reinforced concrete.

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.

Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

Science.gov (United States)

McGinley, Cian; Bishop, David J

2016-12-01

McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βm in vitro ), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βm in vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O 2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

Resurrecting ancestral genes in bacteria to interpret ancient biosignatures

Science.gov (United States)

Kacar, Betul; Guy, Lionel; Smith, Eric; Baross, John

2017-11-01

beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the laboratory, which we term palaeophenotype reconstruction. This article is part of the themed issue 'Reconceptualizing the origins of life'.

Transition Metal Ions Enable the Transition from Electrospun Prolamin Protein Fibers to Nitrogen-Doped Freestanding Carbon Films for Flexible Supercapacitors.

Science.gov (United States)

Wang, Yixiang; Yang, Jingqi; Du, Rongbing; Chen, Lingyun

2017-07-19

Flexible carbon ultrafine fibers are highly desirable in energy storage and conversion devices. Our previous finding showed that electrospun hordein/zein fibers stabilized by Ca 2+ were successfully transferred into nitrogen-doped carbon ultrafine fibers for supercapacitors. However, their relatively brittle nature needed to be improved. Inspired by this stabilizing effect of Ca 2+ , in this work, four transition metal divalent cations were used to assist the formation of flexible hordein/zein-derived carbon ultrafine fibers. Without alteration of the electrospinnability, adequate amounts of zinc acetate and cobalt acetate supported the fibrous structure during pyrolysis. This resulted in flexible freestanding carbon films consisting of well-defined fibers with nitrogen-doped graphitic layers and hierarchical pores. These carbon films were easily cut into small square pieces and directly applied as working electrode in the three-electrode testing system without the need for polymer binders or conducting agents. Notably, the hz-Zn0.3-p electrode, synthesized with 0.3 mol/L Zn 2+ and post-acid treatment, exhibited a specific capacitance of 393 F/g (at 1 A/g), a large rate capability (72.3% remained at 20 A/g), and a capacitance retention of ∼98% after 2000 charging-discharging cycles at 10 A/g. These superior electrochemical properties were attributed to the synergistic effects of the well-developed graphitic layers induced by Zn 2+ , the nitrogen-decorated carbon structure, and the interconnected channels generated by HCl treatment. This research advances potential applications for prolamin proteins as nitrogen-containing raw materials in developing carbon structures for high-performance supercapacitors.

Exploring possible mechanisms of action for the nanotoxicity and protein binding of decorated nanotubes: interpretation of physicochemical properties from optimal QSAR models

International Nuclear Information System (INIS)

Esposito, Emilio Xavier; Hopfinger, Anton J.; Shao, Chi-Yu; Su, Bo-Han; Chen, Sing-Zuo; Tseng, Yufeng Jane

2015-01-01

Carbon nanotubes have become widely used in a variety of applications including biosensors and drug carriers. Therefore, the issue of carbon nanotube toxicity is increasingly an area of focus and concern. While previous studies have focused on the gross mechanisms of action relating to nanomaterials interacting with biological entities, this study proposes detailed mechanisms of action, relating to nanotoxicity, for a series of decorated (functionalized) carbon nanotube complexes based on previously reported QSAR models. Possible mechanisms of nanotoxicity for six endpoints (bovine serum albumin, carbonic anhydrase, chymotrypsin, hemoglobin along with cell viability and nitrogen oxide production) have been extracted from the corresponding optimized QSAR models. The molecular features relevant to each of the endpoint respective mechanism of action for the decorated nanotubes are also discussed. Based on the molecular information contained within the optimal QSAR models for each nanotoxicity endpoint, either the decorator attached to the nanotube is directly responsible for the expression of a particular activity, irrespective of the decorator's 3D-geometry and independent of the nanotube, or those decorators having structures that place the functional groups of the decorators as far as possible from the nanotube surface most strongly influence the biological activity. These molecular descriptors are further used to hypothesize specific interactions involved in the expression of each of the six biological endpoints. - Highlights: • Proposed toxicity mechanism of action for decorated nanotubes complexes • Discussion of the key molecular features for each endpoint's mechanism of action • Unique mechanisms of action for each of the six biological systems • Hypothesized mechanisms of action based on QSAR/QNAR predictive models

Exploring possible mechanisms of action for the nanotoxicity and protein binding of decorated nanotubes: interpretation of physicochemical properties from optimal QSAR models

Energy Technology Data Exchange (ETDEWEB)

Esposito, Emilio Xavier, E-mail: emilio@exeResearch.com [exeResearch, LLC, 32 University Drive, East Lansing, MI 48823 (United States); The Chem21 Group, Inc., 1780 Wilson Drive, Lake Forest, IL 60045 (United States); Hopfinger, Anton J., E-mail: hopfingr@gmail.com [The Chem21 Group, Inc., 1780 Wilson Drive, Lake Forest, IL 60045 (United States); College of Pharmacy MSC09 5360, 1 University of New Mexico, Albuquerque, NM, 87131 (United States); Shao, Chi-Yu [Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan (China); Su, Bo-Han [Department of Computer Science and Information Engineering, National Taiwan University, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan (China); Chen, Sing-Zuo [Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan (China); Tseng, Yufeng Jane, E-mail: yjtseng@csie.ntu.edu.tw [Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan (China); Department of Computer Science and Information Engineering, National Taiwan University, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan (China)

2015-10-01

Carbon nanotubes have become widely used in a variety of applications including biosensors and drug carriers. Therefore, the issue of carbon nanotube toxicity is increasingly an area of focus and concern. While previous studies have focused on the gross mechanisms of action relating to nanomaterials interacting with biological entities, this study proposes detailed mechanisms of action, relating to nanotoxicity, for a series of decorated (functionalized) carbon nanotube complexes based on previously reported QSAR models. Possible mechanisms of nanotoxicity for six endpoints (bovine serum albumin, carbonic anhydrase, chymotrypsin, hemoglobin along with cell viability and nitrogen oxide production) have been extracted from the corresponding optimized QSAR models. The molecular features relevant to each of the endpoint respective mechanism of action for the decorated nanotubes are also discussed. Based on the molecular information contained within the optimal QSAR models for each nanotoxicity endpoint, either the decorator attached to the nanotube is directly responsible for the expression of a particular activity, irrespective of the decorator's 3D-geometry and independent of the nanotube, or those decorators having structures that place the functional groups of the decorators as far as possible from the nanotube surface most strongly influence the biological activity. These molecular descriptors are further used to hypothesize specific interactions involved in the expression of each of the six biological endpoints. - Highlights: • Proposed toxicity mechanism of action for decorated nanotubes complexes • Discussion of the key molecular features for each endpoint's mechanism of action • Unique mechanisms of action for each of the six biological systems • Hypothesized mechanisms of action based on QSAR/QNAR predictive models.

Osmotin, a Pathogenesis-Related Protein

Czech Academy of Sciences Publication Activity Database

Viktorová, J.; Krásný, Lukáš; Kamlar, M.; Nováková, M.; Macková, M.; Macek, T.

2012-01-01

Roč. 13, č. 7 (2012), s. 672-681 ISSN 1389-2037 Grant - others:GA ČR(CZ) GAP501/11/1654; GA ČR(CZ) GA522/09/1693 Program:GA; GA Institutional support: RVO:61388971 Keywords : osmotin * pathogenesis-related proteins * antifungal activity Subject RIV: CE - Biochemistry Impact factor: 2.326, year: 2012

Optic nerve pH and PO2

DEFF Research Database (Denmark)

Pedersen, Daniella B; Stefánsson, Einar; Kiilgaard, Jens Folke

2006-01-01

Earlier studies have demonstrated that carbonic anhydrase inhibitors (CAIs) increase optic nerve oxygen tension (ONPO(2)) in pigs. We hypothesized that the mechanism of this effect was either a CO(2) increase or a pH decrease in tissue and blood. To test this hypothesis we investigated and compared...... how optic nerve pH (ONpH) and ONPO(2) are affected by: (1) carbonic anhydrase inhibition; (2) respiratory acidosis, and (3) metabolic acidosis. We measured ONpH with a glass pH electrode and ONPO(2) with a polarographic oxygen electrode. One of the electrodes was placed in the vitreous cavity 0.5 mm...... over the optic nerve in the eyes of domestic pigs....

Structural studies of human glioma pathogenesis-related protein 1

Energy Technology Data Exchange (ETDEWEB)

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [College of Medicine, Nebraska Medical Center, Omaha, NE 68198-6495 (United States); Koski, Raymond A.; Bonafé, Nathalie [L2 Diagnostics LLC, 300 George Street, New Haven, CT 06511 (United States); College of Medicine, Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

2011-10-01

Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

Bioprecipitation of Calcium Carbonate Crystals by Bacteria Isolated from Saline Environments Grown in Culture Media Amended with Seawater and Real Brine

Directory of Open Access Journals (Sweden)

G. A. Silva-Castro

2015-01-01

Full Text Available The precipitation of calcium carbonate and calcium sulphate by isolated bacteria from seawater and real brine obtained in a desalination plant growth in culture media containing seawater and brine as mineral sources has been studied. However, only bioprecipitation was detected when the bacteria were grown in media with added organic matter. Biomineralization process started rapidly, crystal formation taking place in the beginning a few days after inoculation of media; roughly 90% of total cultivated bacteria showed. Six major colonies with carbonate precipitation capacity dominated bacterial community structure cultivated in heterotrophic platable bacteria medium. Taxonomic identification of these six strains through partial 16S rRNA gene sequences showed their affiliation with Gram-positive Bacillus and Virgibacillus genera. These strains were able to form calcium carbonate minerals, which precipitated as calcite and aragonite crystals and showed bacterial fingerprints or bacteria calcification. Also, carbonic anhydrase activity was observed in three of these isolated bacteria. The results of this research suggest that microbiota isolated from sea water and brine is capable of precipitation of carbonate biominerals, which can occur in situ with mediation of organic matter concentrations. Moreover, calcium carbonate precipitation ability of this microbiota could be of importance in bioremediation of CO2 and calcium in certain environments.

Mechanism of ammonia excretion in the freshwater leech Nephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia

Science.gov (United States)

Quijada-Rodriguez, Alex R.; Treberg, Jason R.

2015-01-01

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)−1·h−1 ammonia and 14.7 ± 1.9 nmol·gFW−1·h−1 urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na+/K+-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na+/K+-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na+/K+-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms. PMID:26180186

Stimulatory effect of Coca-Cola on gastroduodenal HCO3- secretion in rats.

Science.gov (United States)

Sasaki, Y; Aihara, E; Ise, F; Kita, K; Takeuchi, K

2007-10-01

We examined the effect of various carbonated beverages, especially Coca-Cola, on the HCO3- secretion in the rat stomach and duodenum. Under urethane anaesthesia, a chambered stomach or a proximal duodenal loop was perfused with saline, and HCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The amount of CO2 contained in these beverages was about 4-7 g/mL. Coca-Cola topically applied to the mucosa for 10 min significantly increased the HCO3- secretion in both the stomach and the duodenum. The HCO3- response in the duodenum was totally abolished by indomethacin and also partially inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Likewise, the response in the stomach was also markedly inhibited by either acetazolamide or indomethacin. The mucosal application of Coca-Cola increased the PGE2 contents in both the stomach and the duodenum. Other carbonated beverages, such as sparkling water, Fanta Grape or cider, also increased the HCO3- secretion in these tissues. These results suggest that Coca-Cola induces HCO3- secretion in both the stomach and the duodenum, and these responses may be attributable to both the intracellular supply of HCO3- generated via carbonic anhydrase, and endogenous PGs, probably related to the acidic pH of the solution.

Urban energy consumption and related carbon emission estimation: a study at the sector scale

Science.gov (United States)

Lu, Weiwei; Chen, Chen; Su, Meirong; Chen, Bin; Cai, Yanpeng; Xing, Tao

2013-12-01

With rapid economic development and energy consumption growth, China has become the largest energy consumer in the world. Impelled by extensive international concern, there is an urgent need to analyze the characteristics of energy consumption and related carbon emission, with the objective of saving energy, reducing carbon emission, and lessening environmental impact. Focusing on urban ecosystems, the biggest energy consumer, a method for estimating energy consumption and related carbon emission was established at the urban sector scale in this paper. Based on data for 1996-2010, the proposed method was applied to Beijing in a case study to analyze the consumption of different energy resources (i.e., coal, oil, gas, and electricity) and related carbon emission in different sectors (i.e., agriculture, industry, construction, transportation, household, and service sectors). The results showed that coal and oil contributed most to energy consumption and carbon emission among different energy resources during the study period, while the industrial sector consumed the most energy and emitted the most carbon among different sectors. Suggestions were put forward for energy conservation and emission reduction in Beijing. The analysis of energy consumption and related carbon emission at the sector scale is helpful for practical energy saving and emission reduction in urban ecosystems.

Replacement of soybean meal in compound feed by European protein sources : effects on carbon footprint

NARCIS (Netherlands)

Boer, de H.C.; Krimpen, van M.M.; Blonk, H.; Tyszler, M.

2014-01-01

The overall aim was to investigate if soybean products from South American can be replaced by protein sources produced in Europe in a sustainable way. Based on data from literature, and based on the systematics of the FeedPrint programme, the nutritional value and the carbon footprint (CFP) of these

Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

DEFF Research Database (Denmark)

Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen

2008-01-01

, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14)C content from the atmospheric carbon dioxide. The (14)C content of the lens proteins thus reflects the atmospheric content of (14)C when the lens crystallines were formed. Precise radiocarbon...

40 CFR 600.510-12 - Calculation of average fuel economy and average carbon-related exhaust emissions.

Science.gov (United States)

2010-07-01

... and average carbon-related exhaust emissions. 600.510-12 Section 600.510-12 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) ENERGY POLICY FUEL ECONOMY AND CARBON-RELATED EXHAUST EMISSIONS OF... Transportation. (iv) [Reserved] (2) Average carbon-related exhaust emissions will be calculated to the nearest...

Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes

Czech Academy of Sciences Publication Activity Database

Ostatná, Veronika; Černocká, Hana; Kurzatkowska, K.; Paleček, Emil

2012-01-01

Roč. 735, JUL (2012), s. 31-36 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) KJB100040901; GA ČR(CZ) GAP301/11/2055; GA MŠk(CZ) ME09038 Institutional research plan: CEZ:AV0Z50040702 Keywords : protein denaturation * carbon electrodes * edge plane pyrolytic graphite Subject RIV: BO - Biophysics Impact factor: 4.387, year: 2012

A Comprehensive Energy Analysis and Related Carbon Footprint of Dairy Farms, Part 1: Direct Energy Requirements

Directory of Open Access Journals (Sweden)

Giuseppe Todde

2018-02-01

Full Text Available Dairy cattle farms are continuously developing more intensive systems of management which require higher utilization of durable and not-durable inputs. These inputs are responsible of significant direct and indirect fossil energy requirements which are related to remarkable emissions of CO2. This study aims to analyze direct energy requirements and the related carbon footprint of a large population of conventional dairy farms located in the south of Italy. A detailed survey of electricity, diesel and Liquefied Petroleum Gas (LPG consumptions has been carried out among on-farm activities. The results of the analyses showed an annual average fuel consumption of 40 kg per tonne of milk, while electricity accounted for 73 kWh per tonne of milk produced. Expressing the direct energy inputs as primary energy, diesel fuel results the main resource used in on-farm activities, accounting for 72% of the total fossil primary energy requirement, while electricity represents only 27%. Moreover, larger farms were able to use more efficiently the direct energy inputs and reduce the related emissions of carbon dioxide per unit of milk produced, since the milk yield increases with the herd size. The global average farm emissions of carbon dioxide equivalent, due to all direct energy usages, accounted for 156 kg CO2-eq per tonne of Fat and Protein Corrected Milk (FPCM, while farms that raise more than 200 heads emitted 36% less than the average value. In this two-part series, the total energy demand (Part 1 + Part 2 per farm is mainly due to agricultural inputs and fuel consumption, which have the largest quota of the annual requirements for each milk yield class. These results also showed that large size farms held lower CO2-eq emissions when referred to the mass of milk produced.

Prediction of the Ebola Virus Infection Related Human Genes Using Protein-Protein Interaction Network.

Science.gov (United States)

Cao, HuanHuan; Zhang, YuHang; Zhao, Jia; Zhu, Liucun; Wang, Yi; Li, JiaRui; Feng, Yuan-Ming; Zhang, Ning

2017-01-01

Ebola hemorrhagic fever (EHF) is caused by Ebola virus (EBOV). It is reported that human could be infected by EBOV with a high fatality rate. However, association factors between EBOV and host still tend to be ambiguous. According to the "guilt by association" (GBA) principle, proteins interacting with each other are very likely to function similarly or the same. Based on this assumption, we tried to obtain EBOV infection-related human genes in a protein-protein interaction network using Dijkstra algorithm. We hope it could contribute to the discovery of novel effective treatments. Finally, 15 genes were selected as potential EBOV infection-related human genes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effect of tin doping on oxygen- and carbon-related defects in Czochralski silicon

International Nuclear Information System (INIS)

Chroneos, A.; Londos, C. A.; Sgourou, E. N.

2011-01-01

Experimental and theoretical techniques are used to investigate the impact of tin doping on the formation and the thermal stability of oxygen- and carbon-related defects in electron-irradiated Czochralski silicon. The results verify previous reports that Sn doping reduces the formation of the VO defect and suppresses its conversion to the VO 2 defect. Within experimental accuracy, a small delay in the growth of the VO 2 defect is observed. Regarding carbon-related defects, it is determined that Sn doping leads to a reduction in the formation of the C i O i , C i C s , and C i O i (Si I ) defects although an increase in their thermal stability is observed. The impact of strain induced in the lattice by the larger tin substitutional atoms, as well as their association with intrinsic defects and carbon impurities, can be considered as an explanation to account for the above observations. The density functional theory calculations are used to study the interaction of tin with lattice vacancies and oxygen- and carbon-related clusters. Both experimental and theoretical results demonstrate that tin co-doping is an efficient defect engineering strategy to suppress detrimental effects because of the presence of oxygen- and carbon-related defect clusters in devices.

Calculation of the relative chemical stabilities of proteins as a function of temperature and redox chemistry in a hot spring.

Directory of Open Access Journals (Sweden)

Jeffrey M Dick

Full Text Available Uncovering the chemical and physical links between natural environments and microbial communities is becoming increasingly amenable owing to geochemical observations and metagenomic sequencing. At the hot spring known as Bison Pool in Yellowstone National Park, the cooling of the water in the outflow channel is associated with an increase in oxidation potential estimated from multiple field-based measurements. Representative groups of proteins whose sequences were derived from metagenomic data also exhibit an increase in average oxidation state of carbon in the protein molecules with distance from the hot-spring source. The energetic requirements of reactions to form selected proteins used in the model were computed using amino-acid group additivity for the standard molal thermodynamic properties of the proteins, and the relative chemical stabilities of the proteins were investigated by varying temperature, pH and oxidation state, expressed as activity of dissolved hydrogen. The relative stabilities of the proteins were found to track the locations of the sampling sites when the calculations included a function for hydrogen activity that increases with temperature and is higher, or more reducing, than values consistent with measurements of dissolved oxygen, sulfide and oxidation-reduction potential in the field. These findings imply that spatial patterns in the amino acid compositions of proteins can be linked, through energetics of overall chemical reactions representing the formation of the proteins, to the environmental conditions at this hot spring, even if microbial cells maintain considerably different internal conditions. Further applications of the thermodynamic calculations are possible for other natural microbial ecosystems.

Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

Science.gov (United States)

Li, He; Fan, Xinqi; Chen, Xing

2016-02-01

Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

Characterization of the serological biomarkers associated with Sjögren’s syndrome in patients with recalcitrant dry eye disease

Directory of Open Access Journals (Sweden)

Matossian C

2016-07-01

Full Text Available Cynthia Matossian,1,2 Joan Micucci1 1Matossian Eye Associates, Doylestown, PA, USA; 2Department of Ophthalmology, Temple University School of Medicine, Philadelphia, PA, USA Purpose: The purpose was to characterize the biomarkers associated with Sjögren’s syndrome (SS identified in the serological samples of patients with recalcitrant dry eye disease; additionally, the modalities utilized in the treatment of dry eye disease were evaluated for subsets of patients with and without SS. Patients and methods: Data for this retrospective, single-center, pilot study were based on a chart review of 48 sequential patients with recalcitrant dry eye who were evaluated for SS via serological analysis. Data presented include the presence of the autoantibodies identified through the serological biomarker analysis and identification of the concurrent dry eye treatment modalities. Results: Eleven out of 48 patients (23% tested positive for biomarkers associated with SS. Autoantibodies for salivary protein-1, parotid secretory protein 1, and carbonic anhydrase VI, markers associated with the early development of SS, were detected in 91% (ten out of eleven of the patients who tested positive for SS, whereas 27% (three out of eleven of patients tested positive for the traditional SS markers, SS-A and/or SS-B. Common treatment modalities utilized in SS patients included omega-3 supplements (82%, topical cyclosporine (74%, and artificial tear solutions (64%, as compared to omega-3 supplements (80%, hot-mask therapy (77%, and artificial tear solutions (77%, in SS-negative patients. Conclusion: Evaluation for salivary protein-1, parotid secretory protein 1, and carbonic anhydrase VI biomarkers allows for identification of a subset of patients with biomarkers associated with SS that may not be identified through the traditional assessments (SS-A/SS-B. Earlier recognition of SS biomarkers allows for a confirmatory diagnosis and appropriate management of this