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Anhydrase
Kinds of Anhydrase Terms modified by Anhydrase Selected AbstractsCarbonic Anhydrase, Acetazolamide and Helicobacter pylori InfectionHELICOBACTER, Issue 5 2005György M. Buzás No abstract is available for this article. [source] Control of flatfish sperm motility by CO2 and carbonic anhydraseCYTOSKELETON, Issue 3 2003Kazuo Inaba Abstract Sperm motility in flatfishes shows unique characteristics. The flagellar movement either in vivo or in permeabilized models is arrested by the presence of 25,100 mM HCO3,, or by gentle perfusion with CO2 gas. To understand the molecular basis of this property, sperm Triton-soluble proteins and flagellar proteins from several species were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An abundant 29-kDa protein was observed only in flatfish species. Partial amino acid sequences identified this protein as a carbonic anhydrase, an enzyme involved in the interconversion of CO2 and HCO3,. 6-ethoxyzolamide, a specific inhibitor of carbonic anhydrase inhibits sperm motility, especially at low pH. In the case of HCO3, -arrested sperm, the motility is restored by addition of 6-ethoxyzolamide. Taken together, these results suggest that a novel pH/ HCO3, -dependent regulatory mechanism mediated by carbonic anhydrase is involved in the motility control in flatfish sperm. Cell Motil. Cytoskeleton 55:174,187, 2003. © 2003 Wiley-Liss, Inc. [source] Topiramate-induced metabolic acidosis: report of two casesDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 10 2001Chun-hung Ko MRCP FHKAM Medical Officer Two children who presented with symptomatic metabolic acidosis after being put on topiramate (TPM) are reported. The first patient was an 11-year-old male with refractory complex partial epilepsy who was put on TPM for 13 months. He developed hyperventilation 1 week after increasing the dose to 300mg/day. Arterial blood gas revealed hyperchloraemic metabolic acidosis with partial respiratory compensation: pH 7.36, PCO2 27.2 mmHg, bicarbonate 14.9 mEq/L, base excess -8.9 mmol/L. Hyperventilation and acidosis resolved after administration of sodium bicarbonate and reduction of the dose of TPM. The second patient was a female who developed increasing irritability at age 16 months and 21 months, each time associated with introduction of TPM and resolved promptly upon withdrawal of the drug. Venous blood gas taken during the second episode revealed pH 7.34, PCO2 37.4 mmHg, bicarbonate 20.4 mEq/L, base excess -4.2 mmol/L. The predominant mechanism of TPM-induced hyperventilation involves inhibition of carbonic anhydrase at the proximal renal tubule, resulting in impaired proximal bicarbonate reabsorption. The occurrence of hyperpnoea or mental status change in any patient who is on TPM should prompt an urgent blood gas sampling, with correction of the acid-base disturbances accordingly. [source] Atmospheric molding of ionic copolymer MALDI-TOF/MS arrays: A new tool for protein identification/profilingELECTROPHORESIS, Issue 24 2006Alexander Muck Abstract An atmospheric molding protocol has been used to prepare an ionic methacrylate-based copolymer sample support chips for MALDI (pMALDI)-MS by targeting selected groups of various monomers copolymerized during molding, namely, carboxy, sulfo, dimethylalkyamino, and trimethylalkylammonium groups. The new disposable array chips provide analyte-oriented enhancement of protein adsorption to the modified substrates without requiring complicated surface coating or derivatization. The MALDI-MS performance of the new ionic copolymer chips was evaluated for lysozyme, ,-lactoglobulin,A, trypsinogen and carbonic anhydrase,I using washing with solutions prepared in pH or ionic strength steps. On cationic chips, the proteins are washed out at pH lower than their pI values, and on anionic chips at pH higher than their pI values. The ability of the microfabricated pMALDI chip set to selectively adsorb different proteins from real samples and to significantly increase their MS-signal was documented for the transmembrane photosystem,I protein complex from the green alga Chlamydomonas reinhardtii. The proteins were almost exclusively adsorbed according to calculated pI values and grand average of hydropathy (GRAVY) indexes. The new disposable chips reduce manipulation times and increase measurement sensitivity for real-world proteomic samples. The simple atmospheric molding procedure enables additional proteomic operations to be incorporated on disposable MALDI-MS integrated platforms. [source] High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometryELECTROPHORESIS, Issue 6 2005Ya Jin Abstract A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1,M NaOH at 25°C. The recovery of this one-step extraction method was 34,73% for proteins <67,kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within ±0.01,0.10% deviation for all the proteins <36,kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34,ng for cytochrome,c, ,-lactalbumin, myoglobin, ,-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4,29.0,kDa), 340,ng for glyceraldehyde-3-phosphate dehydrogenase (35.6,kDa) and albumin (66.3,kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS. [source] Diversity of the cadmium-containing carbonic anhydrase in marine diatoms and natural watersENVIRONMENTAL MICROBIOLOGY, Issue 2 2007Haewon Park Summary A recent report of a novel carbonic anhydrase (CDCA1) with Cd as its metal centre in the coastal diatom Thalassiosira weissflogii has led us to search for the occurrence of this Cd enzyme (CDCA) in other marine phytoplankton and in the environment. Using degenerate primers designed from the published sequences from T. weissflogii and a putative sequence in the genome of Thalassiosira pseudonana, we show that CDCA is widespread in diatom species and ubiquitous in the environment. All detected genes share more than 64% amino acid identity with the CDCA of T. pseudonana. Analysis of the amino acid sequence of CDCA shows that the putative Cd binding site resembles that of beta-class carbonic anhydrases (CAs). The prevalence of CAs in diatoms that presumably contain Cd at their active site probably reflects the very low concentration of Zn in the marine environment and the difficulty in acquiring inorganic carbon for photosynthesis. The cdca primers developed in this study should be useful for detecting cdca genes in the field, and studying the conditions under which they are expressed. [source] Carbonic Anhydrase Inhibitor Sulthiame Reduces Intracellular pH and Epileptiform Activity of Hippocampal CA3 NeuronsEPILEPSIA, Issue 5 2002Tobias Leniger Summary: ,Purpose: Sulthiame is a carbonic anhydrase (CA) inhibitor with an anticonvulsant effect in the treatment of benign and symptomatic focal epilepsy in children. The aim of the study was to elucidate the mode of action of sulthiame with respect to possible changes of intracellular pH (pHi) that might develop along with sulthiame's anticonvulsant properties. Methods: The effects of sulthiame (a) on pHi of 2,,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymetyl ester (BCECF-AM) loaded CA3 neurones as well as (b) on epileptiform activity (induced by 50 ,M 4-aminopyridine) were compared with those of the CA inhibitors acetazolamide and benzolamide. Results: In the majority of neurons, sulthiame (1.0,1.5 mM; n = 8) as well as the membrane permeant acetazolamide (0.5,1.0 mM; n = 6) reversibly decreased pHi by 0.18 ± 0.05 (SD) and 0.17 ± 0.10 (SD) pH units, respectively, within 10 min. The poor membrane permeant benzolamide (1.0,2.0 mM) had no influence on pHi (n = 8). Sulthiame (1.0,2.5 mM) and acetazolamide (1.0,2.0 mM) reversibly reduced the frequency of action potentials and epileptiform bursts after 10,15 min (n = 9, n = 7), whereas benzolamide (1.0,2.0 mM) had no effect (n = 6). Conclusions: The results suggest that sulthiame acts as a membrane-permeant CA inhibitor whose beneficial effect on epileptiform activity results at least in part from a modest intracellular acidosis of central neurons. [source] Funnel Complexes with CoII and NiII: New Probes into the Biomimetic Coordination Ability of the Calix[6]arene-Based Tris(imidazole) SystemEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 9 2004Olivier Sénèque Abstract The coordination properties of the calix[6]arene-based tris(imidazole) ligand X6Me3Imme3 were further explored with CoII and NiII. This imidazole system stabilizes tetrahedral mononuclear CoII complexes with an exchangeable fourth exogenous ligand (water, alcohol, amide) located at the heart of the hydrophobic calixarene cavity. With a weak donor ligand such as a nitrile, both four-coordinate tetrahedral and five-coordinate trigonal bipyramidal complexes were obtained. The latter contains a second nitrile molecule trans to the included guest nitrile. These complexes were characterized in solution as well as in the solid state. The NiII complexes are square-based pyramidal five-coordinate edifices with a guest nitrile inside the cavity and a water molecule outside. A comparison with previously described ZnII and CuII complexes emphasizes the flexibility of this ligand. A comparison with carbonic anhydrase, a mononuclear zinc enzyme with a tris(histidine) coordination core, shows that X6Me3Imme3 displays many structural features of this enzyme except for the cis coordination of the exogenous ligands. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Proteomic profiling reveals a catalogue of new candidate proteins for human skin agingEXPERIMENTAL DERMATOLOGY, Issue 10 2010Martin Laimer Abstract:, Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from ,young' and ,old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1). [source] Changes in Chemoreflex Characteristics Following Acute Carbonic Anhydrase Inhibition in Humans a RestEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Andrea Vovk The effect of carbonic anhydrase (CA) inhibition with acetazolamide (ACZ, 10 mg kg-1 I.V.) on the peripheral and central chemosensitivity and breathing pattern was investigated in four women and three men aged 25 ± 3 years using a modified version of Read's rebreathing technique. Subjects were exposed to dynamic increases in CO2 in hypoxic and hyperoxic backgrounds during control conditions and following acute CA inhibition. All manoeuvres were repeated twice and averaged for data analysis. The central chemoreflex sensitivities, estimated from the slopes of the ventilatory response to CO2 during hyperoxic rebreathing, increased following acute CA inhibition (control vs. ACZ treatment: 1.87 ± 0.66 vs. 4.07 ± 1.03 l min-1 (mmHg CO2)-1, P < 0.05). The increased slope was reflected by an increase in the rate of rise of tidal volume and breathing frequency. Furthermore with ACZ, there was a left-ward shift of the ventilation vs. end-tidal PCO2 curve during hyperoxic hypercapnia but not hypoxic hypercapnia. The peripheral chemoreflex sensitivity was isolated by subtracting the hyperoxic slope (central only) from the hypoxic slope (central and peripheral). Following ACZ administration, the peripheral chemosensitivity was blunted (control vs. ACZ treatment: 3.66 ± 0.92 vs. 1.33 ± 0.46 l min-1 (mmHg CO2)-1, P < 0.05). In conclusion, acute CA inhibition enhanced the central chemosensitivity to CO2 but diminished the peripheral chemosensitivity. [source] Characterization of carbonic anhydrase from Neisseria gonorrhoeaeFEBS JOURNAL, Issue 6 2001Björn Elleby We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source] Functional demonstration of surface carbonic anhydrase IV activity on rat astrocytesGLIA, Issue 3 2006Nataliya Svichar Abstract Buffering of the brain extracellular fluid is catalyzed by carbonic anhydrase (CA) activity. Whereas the extracellular isoform CA XIV has been localized exclusively to neurons in the brain, and to glial cells in the retina, there has been uncertainty regarding the form or forms of CA on the surface of brain astrocytes. We addressed this issue using physiological methods on cultured and acutely dissociated rat astrocytes. Prior work showed that the intracellular lactate-induced acidification (LIA) of astrocytes is diminished by benzolamide, a poorly permeant, nonspecific CA inhibitor. We demonstrate that pretreatment of astrocytes with phosphatidylinositol-specific phospholipase C (PI-PLC) results in a similar inhibition of the mean LIA (by 66 ± 3%), suggesting that the glycosylphosphatidylinositol-anchored CA IV was responsible. Pretreatment of astrocytes with CA IV inhibitory antisera also markedly reduced the mean LIA in both cultured cortical (by 46 ± 4%) and acutely dissociated hippocampal astrocytes (by 54 ± 8%). Pre-immune sera had no effect. The inhibition produced by PIPLC or CA IV antisera was not significantly less than that by benzolamide, suggesting that the majority of detectable surface CA activity was attributable to CA IV. Thus, our data collectively document the presence of CAIV on the surface of brain astrocytes, and suggest that this is the predominant CA isoform on these cells. © 2005 Wiley-Liss, Inc. [source] CA9 level in renal cyst fluid: a possible molecular diagnosis of malignant tumoursHISTOPATHOLOGY, Issue 7 2009Guorong Li Aims:, The preoperative differentiation of malignant renal cystic tumours from benign lesions is critical, and it remains a common diagnostic problem. The aim was to examine if the Carbonic anhydrase 9 (CA9) level in cyst fluid can provide a molecular diagnosis of malignant cyst. Methods and results:, Twenty-eight patients with a cystic renal mass were included. Fine-needle aspiration was performed to obtain the fluid. Postoperative pathology confirmed that there were 16 cystic renal cell carcinomas. Twelve benign cystic tumours were used as controls. One hundred microlitres of supernatant of cyst fluid was used to measure the CA9 protein level, which was measured by an enzyme-linked immunosorbent assay technique. CA9 was strongly detected and considered as positive in the cyst fluid of all 16 cystic malignant tumours (>1000 pg/ml), whereas its expression was negative in 11/12 benign cystic tumours (<300 pg/ml). The difference in percentages of positive CA9 between malignant and benign renal cystic tumours was significant (P < 0.001). Conclusion:, The fluid of malignant cystic renal tumours contains a high level of CA9 protein. The measurement of CA9 level in cyst fluid may be used as a molecular diagnosis for differentiation between malignant and benign renal cystic masses. [source] Hypoxia-inducible factor-1, in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathwaysINTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Daniel E.B. Swinson Abstract Hypoxia-inducible factor (HIF)-1, is the regulatory subunit of HIF-1 that is stabilized under hypoxic conditions. Under different circumstances, HIF-1, may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF-1, as a prognostic factor. This study evaluated HIF-1, expression in 172 consecutive patients with stage I,IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF-1, nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut-point and 60% as a high positive cut-point. Using the low cut-point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)-9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, p53 (p = 0.008), T-stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl-2 or either N- or overall TMN stage or prognosis. When the high positive cut-point was used, HIF-1, was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP-9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF-1,. The association with TN and squamous-type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF-1,. The prognostic data may reflect a change in the behavior of HIF-1, in increasingly hypoxic environments. © 2004 Wiley-Liss, Inc. [source] Periodicity in proton conduction along a H-bonded chain.INTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 3 2008Application to biomolecules Abstract Molecular complexes are constructed to simulate proton transfer channels of the influenza A virus and of the active site of carbonic anhydrase. These complexes consist of proton donor and acceptor groups connected by a chain of water molecules. Quantum chemical calculations on the methylimidazole(H+)H2OCH3COO, model of the M2 virus channel indicate free translational motion of the water molecule between donor and acceptor, as well as concerted transfer of both H-bond protons. The proton transfer barrier does not depend on the position of the bridged water molecule and varies linearly with the difference of electrostatic potentials between the donor and acceptor. When the water chain is elongated, and with various donor and acceptor models, periodicity appears in the H-bond lengths and the progression of proton transfer in each link. This "wave" is shown to propagate along the chain, as it is driven by the displacement of a single proton. One can thereby estimate the velocity of the proton wave and proton conduction time. Computations are performed to examine the influence of immersing the system within a polarizable medium. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008 [source] Indapamide, a Thiazide-Like Diuretic, Decreases Bone Resorption In VitroJOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2001Agnes Lalande Abstract We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10,4 M IDP and 10,4 M AZ, as well as 10,5 M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10,8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl, cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10,4 M IDP as well as 10,5 M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors. [source] Effect of instrument tuning on the detectabilityof biopolymers in electrospray ionization mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2003Herbert Oberacher Abstract Electrospray ionization mass spectrometry of multiply charged biopolymer ions of different molecular size revealed a strong influence of tuning parameters on their detectability in quadrupole ion trap and triple quadrupole mass spectrometers. Hence, after optimizing the ion optical parameters with the signal of the 4, charge state of (dT)24 (low charge state tuning), a tenfold increase in the signal-to-noise ratio for a mixture of oligodeoxythymidylic acids (n = 12,18) was obtained compared with the results achieved with tune parameters optimized with a synthetic 80-mer oligodeoxynucleotide. By contrast, a detection limit in the upper femtomole region could only be reached for a 104-mer oligodeoxynucleotide utilizing the 24, charge state of the 80-mer (high charge state tuning). The same effect was observed for proteins investigated in the positive ion mode using low and high charge states of cytochrome c and carbonic anhydrase, respectively, for instrument tuning. By comparing the settings for low and high charge state tuning, it became obvious that the most significant difference was observed in the potential applied to the heated metal capillary used to transfer ions from the atmospheric pressure to the vacuum region of the ion source. Taking advantage of the optimized tuning procedure, the molecular mass of a 61 base pair product of polymerase chain reaction was accurately determined by electrospray ionization mass spectrometry on-line interfaced to ion-pair reversed-phase high-performance liquid chromatography. Copyright © 2003 John Wiley & Sons, Ltd. [source] Up-regulation of cerebral carbonic anhydrase by anoxic stress in pigletsJOURNAL OF NEUROCHEMISTRY, Issue 4 2003Antal Nógrádi Abstract The resuscitation of asphyxiated babies is associated with changes in cerebral protein synthesis that can influence the neurological outcome. Insufficient gas exchange results in rapid shifts in extracellular and intracellular pH. Carbonic anhydrase (CA) plays an important role in buffering acute changes in pH in the brain. We investigated whether asphyxia/re-ventilation influences the expression of cerebral CA isoforms (CA-II, CA-III and CA-IV) in anaesthetized newborn pigs. The cerebral cortex, hippocampus, cerebellum and retina were sampled, and prepared for either CA immunohistochemistry or CA immunoblotting from piglets subjected to asphyxia (10 min) followed by 2,4 h of re-ventilation, and also from normoxic controls. The CA immunoreactivity (IR) of all the isoforms studied was weak in the controls, apart from staining of a few oligodendrocytes in the subcortical white matter, some astrocytes in the superficial layer of the cerebral cortex, the cerebellar Purkinje cells and the retinal Müller cells that possessed moderate CA-II IR. However, asphyxia induced a marked increase in the CA IR of all isoforms in all the cerebral regions investigated and the retina after 4 h of survival. The pyramidal cells of the frontal cortex and hippocampus displayed the most conspicuous increase in CA IR. Immunoblotting confirmed increased levels of all the CA isoenzymes. We conclude that raised CA levels after asphyxia may contribute to the compensatory mechanisms that protect against the pathological changes in the neonatal CNS. [source] An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticlesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2005James Chesko Abstract This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2510-2519, 2005 [source] Cyclodextrin complexes of sulfonamide carbonic anhydrase inhibitors as long-lasting topically acting antiglaucoma agentsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2002Francesca Maestrelli Abstract Complexes of several 1,3,4-thiadiazole-2-sulfonamide derivatives possessing strong carbonic anhydrase (CA) inhibitory properties with ,-cyclodextrin and hydroxypropyl-,-cyclodextrin were obtained and characterized. Although the investigated CA inhibitors possessed very powerful inhibitory properties against the two CA isozymes involved in aqueous humor production within the eye, i.e., CA II and CA IV, these compounds were topically ineffective as intraocular pressure (IOP) lowering agents in normotensive/hypertensive rabbits, due to their very low water solubility. On the contrary, the cyclodextrin,sulfonamide complexes proved to be effective and long-lasting IOP lowering agents in the two animal models of glaucoma mentioned above. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2211,2219, 2002 [source] EFFECT OF CARBONIC ANHYDRASE INHIBITORS ON THE INORGANIC CARBON UPTAKE BY PHYTOPLANKTON NATURAL ASSEMBLAGES,JOURNAL OF PHYCOLOGY, Issue 1 2009Jesús M. Mercado The role of carbonic anhydrase (CA) in inorganic carbon acquisition (dissolved inorganic carbon, DIC) was examined in Alboran Sea phytoplankton assemblages. The study area was characterized by a relatively high variability in nutrient concentration and in abundance and taxonomic composition of phytoplankton. Therefore, the relationship between environmental variability and capacity for using HCO3, via external CA (eCA) was examined. Acetazolamide (AZ, an inhibitor of eCA) inhibited the primary productivity (PP) in 50% of the samples, with inhibition percentages ranging from 13% to 60%. The AZ effect was more prominent in the samples that exhibited PP >1 mg C · m,3 · h,1, indicating that the contribution of eCA to the DIC photosynthetic flux was irrelevant at low PP. The inhibition of primary productivity by AZ was significantly correlated to the abundance of diatoms. However, there was no a relationship between AZ effect and CO2 partial pressure (pCO2) or nutrient concentration, indicating that the variability in the PP percentage supported by eCA was mainly due to differences in taxonomic composition of the phytoplankton assemblages. Ethoxyzolamide (EZ, an inhibitor of both external and internal CA) affected 13 of 14 analyzed samples, with PP inhibition percentages varying from 50% to 95%. The effects of AZ and EZ were partially reversed by doubling DIC concentration. These results imply that CA activity (external and/or internal) was involved in inorganic carbon acquisition in most the samples. However, EZ effect was not correlated with pCO2 or taxonomic composition of the phytoplankton. [source] PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA, HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA,JOURNAL OF PHYCOLOGY, Issue 6 2003Dinghui Zou The mechanism of inorganic carbon (Ci) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO2 production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion-exchanger inhibitor 4,4,-diisothiocyano-stilbene-2,2,-disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO3, as a source of Ci for its photosynthesis via the mediation of the external CA. CO2 concentrations in the culture media affected its photosynthetic properties. A high level of CO2 (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO2 level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO2 was a reduction in the photosynthetic CO2 affinity. Decreased activity of the external CA in the high CO2 grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO2 -limited photosynthesis with the half-saturating concentration of Ci exceeding that of seawater, did not operate active HCO3, uptake but used it via the extracellular CA for its photosynthetic carbon fixation. [source] Prediction of Carbonic Anhydrase Activation by Tri-/Tetrasubstituted-pyridinium-azole Compounds: A Computational Approach using Novel Topochemical DescriptorMOLECULAR INFORMATICS, Issue 7 2004Sanjay Bajaj Abstract A novel highly discriminating adjacency-cum-distance based topochemical descriptor, termed as Superadjacency topochemical index, has been derived and its discriminating power investigated with regard to activation of Carbonic anhydrase (CA) isozyme-I by tri-/tetrasubstituted-pyridinium-azole compounds. The new index is not only sensitive to the presence of heteroatoms but also overcomes the problem of degeneracy of many topological descriptors. The discriminating power of Superadjacency topochemical index was found to be far superior when compared with that of distance based Wiener,s index and adjacency based Molecular connectivity index. The values of Wiener,s index, path-one Molecular connectivity index and Superadjacency topochemical index of each of the 42 substituted-pyridinium-azole compounds comprising the dataset were computed. Resultant data was analyzed and suitable models developed after identification of the active ranges. Subsequently, a biological activity was assigned to each of the compounds involved in the dataset using these models, which was then compared with the reported activation constants for Carbonic Anhydrase isozyme-I. Excellent correlations were observed between the activation constants of CA isozyme-I and all the topological/topochemical descriptors. The overall accuracy of prediction was about 91% for models based upon both Molecular connectivity index as well as Wiener,s index, and 96% for model based upon Superadjacency topochemical index. Surprisingly, the accuracy of prediction in the active range was found to be 100% in all the models. Thus the proposed index offers a vast potential for structure-activity/structure-property studies. [source] Colonic and small-intestinal phenotypes in gastric cancers: Relationships with clinicopathological findingsPATHOLOGY INTERNATIONAL, Issue 10 2005Tsutomu Mizoshita The clinicopathological significance of colonic and small-intestinal phenotypes has hitherto remained unclear in gastric cancers. The purpose of the present study was therefore to examine 86 gastric carcinomas histologically and phenotypically using several phenotypic markers, including colon-specific carbonic anhydrase 1 (CA1) and sucrase as small-intestine specific marker. Of 86 gastric cancers, sucrase and CA1 expression was observed in 12 (14.0%) and only in two cases (2.3%), respectively, associated with other intestinal markers such as villin and mucin core protein (MUC)2. In the sucrase cases, expression appeared independent of the stage. However, CA1 expression was observed only in two advanced cases. No association was observed between colonic and small-intestinal phenotypes, and lymph node metastasis and postoperative survival in the advanced gastric cancer cases with intestinal phenotypic expression. Cdx2 appeared to be linked to upregulation of both CA1 and sucrase. In conclusion, the data suggest that colonic phenotype occurs rarely in gastric carcinogenesis. Colonic and small-intestinal phenotypes appear with expression of several intestinal phenotypic markers under the control of Cdx2 and presumably other related transcription factors. [source] Composition and activity of external carbonic anhydrase of microalgae from karst lakes in ChinaPHYCOLOGICAL RESEARCH, Issue 2 2008Yanyou Wu SUMMARY The species composition, the net photosynthetic O2 evolution rate and the activity of external carbonic anhydrase (CA) of microalgae from three reservoirs were studied. Carbonic anhydrase activity had a significant positive correlation with the density of Cyanobacteria in Lake Aha. Microalgae's carbonic anhydrase activity in Lakes Baihua and Hongfeng was related to the density of Chlorophyceae. The species abundances of microalgae in Lake Aha, Lake Baihua, and Lake Hongfeng were different. A relationship between carbonic anhydrase activity and net photosynthetic O2 evolution rate had also been established. Algae with external CA influenced the algal productivity. These results demonstrate the role of external CA in facilitating the availability of CO2 that limits the photosynthesis of microalgae in karst lakes in China. [source] Carbonic anhydrases in plants and algaePLANT CELL & ENVIRONMENT, Issue 2 2001J. V. Moroney ABSTRACT Carbonic anhydrases catalyse the reversible hydration of CO2, increasing the interconversion between CO2 and HCO3, + H+ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated ,-, ,-and ,-CA. Animals have only the ,-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C3 dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases. [source] Naturally low carbonic anhydrase activity in C4 and C3 plants limits discrimination against C18OO during photosynthesisPLANT CELL & ENVIRONMENT, Issue 9 2000J. S. Gillon ABSTRACT The 18O content of CO2 is a powerful tracer of photosynthetic activity at the ecosystem and global scale. Due to oxygen exchange between CO2 and 18O-enriched leaf water and retrodiffusion of most of this CO2 back to the atmosphere, leaves effectively discriminate against 18O during photosynthesis. Discrimination against 18O (,18O) is expected to be lower in C4 plants because of low ci and hence low retrodiffusing CO2 flux. C4 plants also generally show lower levels of carbonic anhydrase (CA) activities than C3 plants. Low CA may limit the extent of 18O exchange and further reduce ,18O. We investigated CO2,H2O isotopic equilibrium in plants with naturally low CA activity, including two C4 (Zea mays, Sorghum bicolor) and one C3 (Phragmites australis) species. The results confirmed experimentally the occurrence of low ,18O in C4, as well as in some C3, plants. Variations in CA activity and in the extent of CO2,H2O isotopic equilibrium (,eq) estimated from on-line measurements of ,18O showed large range of 0,100% isotopic equilibrium (,eq= 0,1). This was consistent with direct estimates based on assays of CA activity and measurements of CO2 concentrations and residence times in the leaves. The results demonstrate the potential usefulness of ,18O as indicator of CA activity in vivo. Sensitivity tests indicated also that the impact of ,eq< 1 (incomplete isotopic equilibrium) on 18O of atmospheric CO2 can be similar for C3 and C4 plants and in both cases it increases with natural enrichment of 18O in leaf water. [source] The structure and evolution of the murine inhibitor of carbonic anhydrase: A member of the transferrin superfamilyPROTEIN SCIENCE, Issue 9 2010Brian E. Eckenroth Abstract The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 Å structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two ,-, domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse ,-turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N-lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N-lobe of lactoferrin may be in the process of eliminating its iron binding function. [source] Crystal structure of E. coli ,,carbonic anhydrase, an enzyme with an unusual pH,dependent activityPROTEIN SCIENCE, Issue 5 2001Jeff D. Cronk CA, carbonic anhydrase; ECCA, Escherichia coli ,-carbonic anhydrase; PPCA, Porphyridium purpureum ,-carbonic anhydrase; PSCA, Pisum sativum ,-carbonic anhydrase; EXAFS, extended X-ray absorption fine structure spectroscopy; MAD, multiwavelength anomalous dispersion Abstract Carbonic anhydrases fall into three distinct evolutionary and structural classes: ,, ,, and ,. The ,-class carbonic anhydrases (,-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a ,-CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the ,-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some ,-CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a ,-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other ,-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates. [source] Prevalidation of potential protein biomarkers in toxicology using iTRAQÔ reagent technologyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2007Matthias Glückmann Abstract Today, toxicoproteomics still relies mainly on 2-DE followed by MS for detection and identification of proteins, which might characterize a certain state of disease, indicate toxicity or even predict carcinogenicity. We utilized the classical 2-DE/MS approach for the evaluation of early protein biomarkers which are predictive for chemically induced hepatocarcinogenesis in rats. We were able to identify statistically significantly deregulated proteins in N -nitrosomorpholine exposed rat liver tissue. Based on literature data, biological relevance in the early molecular process of hepatocarcinogenicity could be suggested for most of these potential biomarkers. However, in order to ensure reliable results and to create the prerequisites necessary for integration in routine toxicology studies in the future, these protein expression patterns need to be prevalidated using independent technology platforms. In the current study, we evaluated the usefulness of iTRAQÔ reagent technology (Applied Biosystems, Framingham, USA), a recently introduced MS-based protein quantitation method, for verification of the 2-DE/MS biomarkers. In summary, the regulation of 26 2-DE/MS derived protein biomarkers could be verified. Proteins like HSP 90-beta, annexin A5, ketohexokinase, N -hydroxyarylamine sulfotransferase, ornithine aminotransferase, and adenosine kinase showed highly comparable fold changes using both proteomic quantitation strategies. In addition, iTRAQ analysis delivered further potential biomarkers with biological relevance to the processes of hepatocarcinogenicity: e.g. placental form of glutathione S-transferase (GST-P), carbonic anhydrase, and aflatoxin B1 aldehyde reductase. Our results show both the usefulness of iTRAQ reagent technology for biomarker prevalidation as well as for identification of further potential marker proteins, which are indicative for liver hepatocarcinogenicity. [source] | |||||||||||||||||||||||||||||||