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Sample records for anhydrase ix expression

  1. Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms.

    PubMed

    Syrjänen, Leo; Luukkaala, Tiina; Leppilampi, Mari; Kallioinen, Matti; Pastorekova, Silvia; Pastorek, Jaromir; Waheed, Abdul; Sly, William S; Parkkila, Seppo; Karttunen, Tuomo

    2014-09-01

    Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours.

  2. Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models

    PubMed Central

    Chen, Liu Qi; Howison, Christine M.; Spier, Catherine; Stopeck, Alison T.; Malm, Scott W.; Pagel, Mark D.; Baker, Amanda F.

    2015-01-01

    The expression of carbonic anhydrase (CA IX) and it’s relation to acidosis in lymphomas has not been widely studied. We investigated the protein expression of CA IX in a human B-cell lymphoma tissue microarray, and in Raji, Ramos, and Granta 519 lymphoma cell lines and tumor models, while also investigating the relation with hypoxia. An imaging method, acidoCEST MRI, was used to estimate lymphoma xenograft extracellular pH (pHe). Our results showed that clinical lymphoma tissues and cell line models in vitro and in vivo had moderate CA IX expression. Although in vitro studies showed that CA IX expression was induced by hypoxia, in vivo studies did not show this correlation. Untreated lymphoma xenograft tumor pHe had acidic fractions, and an Acidity Score was qualitatively correlated with CA IX expression. Therefore, CA IX is expressed in B-cell lymphomas and is qualitatively correlated with extracellular acidosis in xenograft tumor models. PMID:25130478

  3. Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX.

    PubMed

    Garousi, Javad; Honarvar, Hadis; Andersson, Ken G; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Löfblom, John; Frejd, Fredrik Y; Tolmachev, Vladimir

    2016-11-07

    Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant tumors, for patients' stratification for CAIX-targeted therapies, and for discrimination of primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small affinity proteins based on a nonimmunoglobulin scaffold. In this study, we compared imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts and competing for the same epitope on CAIX, but having different binding paratopes. Four variants were labeled using residualizing (99m)Tc and nonresidualizing (125)I labels. All radiolabeled variants demonstrated high-affinity detection of CAIX-expressing cell line SK-RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. (125)I-labeled conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity concentration in blood compared with (99m)Tc-labeled counterparts. Although all variants cleared rapidly from blood and nonspecific compartments, there was noticeable difference in their biodistribution. The best variant for imaging of expression of CAIX in disseminated cancer was (99m)Tc-(HE)3-ZCAIX:2 providing tumor uptake of 16.3 ± 0.9% ID/g and tumor-to-blood ratio of 44 ± 7 at 4 h after injection. For primary renal cell carcinoma, the most promising imaging candidate was (125)I-ZCAIX:4 providing tumor-kidney ratio of 2.1 ± 0.5. In conclusion, several clones of scaffold proteins should be evaluated to select the best variant for development of an imaging probe with optimal sensitivity for the intended application.

  4. Dexamethasone downregulates expression of carbonic anhydrase IX via HIF-1α and NF-κB-dependent mechanisms

    PubMed Central

    Simko, Veronika; Takacova, Martina; Debreova, Michaela; Laposova, Katarina; Ondriskova-Panisova, Elena; Pastorekova, Silvia; Csaderova, Lucia; Pastorek, Jaromir

    2016-01-01

    Dexamethasone is a synthetic glucocorticoid frequently used to suppress side-effects of anticancer chemotherapy. In the present study, we showed that dexamethasone treatment leads to concentration-dependent downregulation of cancer-associated marker, carbonic anhydrase IX (CA IX), at the level of promoter activity, mRNA and protein expression in 2D and 3D cancer cell models. The effect of dexamethasone on CA IX expression under hypoxic conditions is predominantly mediated by impaired transcriptional activity and decreased protein level of the main hypoxic transcription factor HIF-1α. In addition, CA9 downregulation can be caused by protein-protein interactions between activated glucocorticoid receptors, major effectors of glucocorticoid action, and transcription factors that trigger CA9 transcription (e.g. AP-1). Moreover, we identified a potential NF-κB binding site in the CA9 promoter and propose the involvement of NF-κB in the dexamethasone-mediated inhibition of CA9 transcription. As high level of CA IX is often linked to aggressive tumor behavior, poor prognosis and chemo- and radiotherapy resistance, uncovering its reduction after dexa-methasone treatment and implication of additional regulatory mechanisms can be relevant for the CA IX-related clinical applications. PMID:27431580

  5. Induction by hypoxia combined with low glucose or low bicarbonate and high posttranslational stability upon reoxygenation contribute to carbonic anhydrase IX expression in cancer cells.

    PubMed

    Rafajová, Monika; Zatovicová, Miriam; Kettmann, Richard; Pastorek, Jaromír; Pastoreková, Silvia

    2004-04-01

    Hypoxia is an important factor of tumor microenvironment that significantly influences behaviour of tumor cells via activation of genes whose products are involved in adaptation to hypoxic stress, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT-1). Carbonic anhydrase IX (CA IX) is one of the most strongly hypoxia-inducible proteins with potential value as an intrinsic marker of hypoxia. However, intratumoral distribution of CA IX only partially overlaps with distribution of VEGF and GLUT-1 indicating that regulation of CA IX differs from the regulation of other hypoxic markers. Therefore, we analysed CA IX expression in response to hypoxia combined with other stresses, and determined the stability of CA IX protein upon reoxygenation using HeLa cells as a model. We found that both hypoxia-induced transcription and CA IX protein level are further increased by reduced glucose or bicarbonate concentrations. Post-translational stability of CA IX was assessed by monitoring the quantity of biotinylated protein extracted at different time points from the cells labelled immediately after shift to reoxygenation. CA IX protein half-life in reoxygenated cells was 38 h and was independent of the duration of the foregoing hypoxia. This finding has potential implications for interpretation of clinical data as it suggests that CA IX expression may detect not only actually hypoxic tumor regions, but also the regions affected by hypoxia and adverse microenvironmental stresses before biopsy or tumor removal.

  6. Roles of Carbonic Anhydrase IX in Development of Pancreatic Cancer.

    PubMed

    Li, Yuji; Dong, Ming; Sheng, Weiwei; Huang, Longping

    2016-04-01

    The aim of this study was to study the effects of carbonic anhydrase IX (CA IX) towards the invasion and metastasis of pancreatic cancer. The expressions of CA IX in 58 cases of pancreatic cancer and paired paracancerous normal tissues, obtained from 2005 to 2012 in the first Affiliated Hospital of China Medical University, were detected, as well as its expressions in different pancreatic cancer cell lines, aiming to detect the impacts of CA IX silencing towards the invasion and metastasis of pancreatic cancer cells. The CA IX expressions in 58 pancreatic cancer cases were higher than those in the paired paracancerous normal tissues (P < 0.01), and positively correlated with the tumor size and the UICC staging UICC (P < 0.05), the multivariate analysis showed that the high expression of CA IX was the independent risk factor towards the prognosis of pancreatic cancer (P < 0.05). The CA IX was highly expressed in AxPC-1 and Miapaca-2, and the interference effects were significant. CA IX silencing could significantly inhibit the invasion and metastasis of AxPC-1 and Miapaca. We support a pro-tumor role of CA IX in the development and progression of pancreatic cancer.

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

    SciTech Connect

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

    2011-05-01

    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.

  8. Carbonic anhydrase expression in kidney and renal cancer: implications for diagnosis and treatment.

    PubMed

    Oosterwijk, Egbert

    2014-01-01

    Four different carbonic anhydrases are expressed in the human nephron, the functional unit of the kidney. These are specifically expressed in different nephron segments, emphasizing the critical role carbonic anhydrases play in maintaining the homeostasis of this crucial organ.Whereas the localization of carbonic anhydrases in the kidney has been long established, interest in carbonic anhydrases has increased dramatically for renal cancer, in particular for the clear cell variant of renal cell carcinoma (ccRCC) because carbonic anhydrase IX is specifically expressed in ccRCC. Therefore carbonic anhydrase IX is being studied as potential diagnostic and therapeutic target, despite carbonic anhydrase IX expression in non-renal tissues.

  9. Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

    PubMed

    Klier, Michael; Jamali, Somayeh; Ames, Samantha; Schneider, Hans-Peter; Becker, Holger M; Deitmer, Joachim W

    2016-01-01

    Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

  10. A New Peptide Ligand for Targeting Human Carbonic Anhydrase IX, Identified through the Phage Display Technology

    PubMed Central

    Askoxylakis, Vasileios; Garcia-Boy, Regine; Rana, Shoaib; Krämer, Susanne; Hebling, Ulrike; Mier, Walter; Altmann, Annette; Markert, Annette; Debus, Jürgen; Haberkorn, Uwe

    2010-01-01

    Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. Methods Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. Results In vitro binding experiments of 125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. Conclusions These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX. PMID:21209841

  11. Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients

    PubMed Central

    Kalavska, Katarina; Chovanec, Michal; Zatovicova, Miriam; Takacova, Martina; Gronesova, Paulina; Svetlovska, Daniela; Baratova, Magdalena; Miskovska, Vera; Obertova, Jana; Palacka, Patrik; Rajec, Jan; Sycova-Mila, Zuzana; Cierna, Zuzana; Kajo, Karol; Spanik, Stanislav; Babal, Pavel; Mardiak, Jozef; Pastorekova, Silvia; Mego, Michal

    2016-01-01

    Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics. PMID:27698832

  12. Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients.

    PubMed

    Kalavska, Katarina; Chovanec, Michal; Zatovicova, Miriam; Takacova, Martina; Gronesova, Paulina; Svetlovska, Daniela; Baratova, Magdalena; Miskovska, Vera; Obertova, Jana; Palacka, Patrik; Rajec, Jan; Sycova-Mila, Zuzana; Cierna, Zuzana; Kajo, Karol; Spanik, Stanislav; Babal, Pavel; Mardiak, Jozef; Pastorekova, Silvia; Mego, Michal

    2016-10-01

    Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.

  13. Circulating Carbonic Anhydrase IX and Antiangiogenic Therapy in Breast Cancer

    PubMed Central

    Brown-Glaberman, Ursa; Marron, Marilyn; Chalasani, Pavani; Livingston, Robert; Iannone, Maria; Specht, Jennifer; Stopeck, Alison T.

    2016-01-01

    Introduction. Carbonic anhydrase IX (CAIX) is a hypoxia regulated metalloenzyme integral to maintaining cellular pH. Increased CAIX expression is associated with poor prognosis in breast cancer. To explore CAIX as a biomarker for breast cancer therapies, we measured plasma CAIX levels in healthy control subjects and in breast cancer patients. Methods. In control subjects we evaluated plasma CAIX stability via commercially available ELISA. We then similarly quantified plasma CAIX levels in (1) locally advanced breast cancer (LABC) patients treated with neoadjuvant paclitaxel + sunitinib (T + S) followed by doxorubicin and cyclophosphamide (AC); (2) metastatic breast cancer (MBC) patients treated with systemic chemotherapy. Results. Plasma CAIX levels were stable at room temperature for at least 48 hours in control subjects. Mean baseline plasma CAIX levels were lower in controls compared to patients with LABC or MBC. In LABC, CAIX levels rose significantly in response to administration of antiangiogenic therapy (T + S) (p = 0.02) but not AC (p = 0.37). In patients with MBC treated without an antiangiogenic agent CAIX levels did not change with therapy. Conclusions. Our results suggest that CAIX may be an easily obtained, stable measure of tumor associated hypoxia as well as a useful pharmacodynamic biomarker for antiangiogenic therapy. PMID:26941473

  14. Optimization of a Novel Peptide Ligand Targeting Human Carbonic Anhydrase IX

    PubMed Central

    Rana, Shoaib; Nissen, Felix; Marr, Annabell; Markert, Annette; Altmann, Annette; Mier, Walter; Debus, Juergen; Haberkorn, Uwe; Askoxylakis, Vasileios

    2012-01-01

    Background Carbonic anhydrase IX (CA IX) is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1) was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. Methodology/Principal Findings Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy) was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. Conclusions Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of the ligand in

  15. Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells

    SciTech Connect

    Vordermark, Dirk . E-mail: vordermark_d@klinik.uni-wuerzburg.de; Kaffer, Anja; Riedl, Susanne; Katzer, Astrid; Flentje, Michael

    2005-03-15

    Purpose: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. Methods and materials: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. Results: Hypoxia-induced CA IX protein expression was seen after >6 h at {<=}5% O{sub 2}, and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. Conclusion: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1{alpha}. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro

  16. Recent Developments in Targeting Carbonic Anhydrase IX for Cancer Therapeutics

    PubMed Central

    McDonald, Paul C.; Winum, Jean-Yves; Supuran, Claudiu T.; Dedhar, Shoukat

    2012-01-01

    Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme that is overexpressed by cancer cells from many tumor types, and is a component of the pH regulatory system invoked by these cells to combat the deleterious effects of a high rate of glycolytic metabolism. CAIX functions to help produce and maintain an intracellular pH (pHi) favorable for tumor cell growth and survival, while at the same time participating in the generation of an increasingly acidic extracellular space, facilitating tumor cell invasiveness. Pharmacologic interference of CAIX catalytic activity using monoclonal antibodies or CAIX-specific small molecule inhibitors, consequently disrupting pH regulation by cancer cells, has been shown recently to impair primary tumor growth and metastasis. Many of these agents are in preclinical or clinical development and constitute a novel, targeted strategy for cancer therapy. PMID:22289741

  17. Carbonic anhydrase IX-directed immunoliposomes for targeted drug delivery to human lung cancer cells in vitro.

    PubMed

    Wong, Blenda Chi Kwan; Zhang, Hongqi; Qin, Ling; Chen, Hubiao; Fang, Chen; Lu, Aiping; Yang, Zhijun

    2014-01-01

    Targeted drug delivery to cancer cells by use of antibody-conjugated liposomes (immunoliposomes) has attracted considerable interest in recent years. Despite increasing efforts in developing immunoliposomes as drug carriers, the investigation of useful tumor-associated antigen targets is far from complete. Carbonic anhydrase IX (CA IX) is a cell surface antigen characterized by hypoxia-induced expression in many solid tumors. This study investigated the feasibility of CA IX-directed immunoliposomes for targeted delivery of docetaxel to human lung cancer cells in vitro. Docetaxel-loaded immunoliposomes targeting CA IX were developed with an encapsulation efficiency of 84.4±3.9% and an average particle size of 143.9±11.1 nm. Using fluorescence-based flow cytometry, the in vitro binding activity of the immunoliposomes was found to be significantly higher (by 1.65-fold) than that of the nontargeted liposomes in CA IX-positive lung cancer cells, whereas no such difference was observed between the two groups when CA IX was not expressed. Furthermore, immunoliposomal docetaxel exhibited the strongest growth inhibitory effect against CA IX-positive lung cancer cells when compared with nontargeted liposomal docetaxel or free docetaxel solution. These data suggested that CA IX-directed immunoliposomes could serve as a promising drug delivery system for targeted killing of lung cancer cells.

  18. Chapter 13: Carbonic Anhydrase IX as an Imaging and Therapeutic Target for Tumors and Metastases

    PubMed Central

    Tafreshi, Narges K.; Lloyd, Mark C.; Bui, Marilyn M.; Gillies, Robert J.; Morse, David L.

    2014-01-01

    Carbonic anhydrase IX (CAIX) which is a zinc containing metalloprotein, efficiently catalyzes the reversible hydration of carbon dioxide. It is constitutively up-regulated in several cancer types and has an important role in tumor progression, acidification and metastasis. High expression of CAIX generally correlates with poor prognosis and is related to a decrease in the disease-free interval following successful therapy. Therefore, it is considered as a prognostic indicator in oncology. In this review, we describe CAIX regulation and its role in tumor hypoxia, acidification and metastasis. In addition, the molecular imaging of CAIX and its potential for use in cancer detection, diagnosis, staging, and for use in following therapy response is discussed. Both antibodies and small molecular weight compounds have been used for targeted imaging of CAIX expression. The use of CAIX expression as an attractive and promising candidate marker for systemic anticancer therapy is also discussed. PMID:24146382

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

    PubMed

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

    2015-02-15

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

  20. Saccharin: a Lead Compound for Structure-Based Drug Design of Carbonic Anhydrase IX Inhibitors

    PubMed Central

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

    2015-01-01

    Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition. PMID:25614109

  1. Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.

    PubMed

    Alterio, Vincenzo; Hilvo, Mika; Di Fiore, Anna; Supuran, Claudiu T; Pan, Peiwen; Parkkila, Seppo; Scaloni, Andrea; Pastorek, Jaromir; Pastorekova, Silvia; Pedone, Carlo; Scozzafava, Andrea; Monti, Simona Maria; De Simone, Giuseppina

    2009-09-22

    Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the alpha-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical alpha-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO(2) hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated alpha-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

  2. CREB is a key negative regulator of carbonic anhydrase IX (CA9) in gastric cancer.

    PubMed

    Wang, Guanqiao; Cheng, Zhenguo; Liu, Funan; Zhang, Hongyan; Li, Jiabin; Li, Feng

    2015-07-01

    Carbonic anhydrase IX(CA9)is a member of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide, and plays a key role in the regulation of pH. Although a large number of studies have shown that CA9 is strongly up-regulated by HIF1-α, little is known about the negative regulation mechanism of CA9 in cancer cells. Here we find that CREB is a key negative regulator of CA9 in gastric cancer. Over-expression of CREB can significantly repress the expression of CA9. Treating with anisomycin (ANS), an activator of p38, the phosphorylation and nuclear translocation of CREB are both promoted, while the transcription of CA9 is repressed. Besides, our results firstly identify that CREB can recruit SIRT1 (class III HDACS) by adaptor protein p300, then repress the expression of CA9. These findings may contribute to understand the negative regulation mechanisms of CA9 in gastric cancer.

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

    PubMed

    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.

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

    PubMed

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

    2016-07-14

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

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

    PubMed

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

    2014-01-08

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

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

    DOE PAGES

    Pinard, Melissa A.; Aggarwal, Mayank; Mahon, Brian P.; ...

    2015-09-23

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2to HCO3$-$, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereasmore » the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, anRcryst of 18.0% and anRfree of 21.2%. Finally, the binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.« less

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

    PubMed Central

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

    2015-01-01

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3 −, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an R cryst of 18.0% and an R free of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX. PMID:26457530

  8. Carbonic anhydrase enzymes II, VII, IX and XII in colorectal carcinomas

    PubMed Central

    Viikilä, Pia; Kivelä, Antti J; Mustonen, Harri; Koskensalo, Selja; Waheed, Abdul; Sly, William S; Pastorek, Jaromir; Pastorekova, Silvia; Parkkila, Seppo; Haglund, Caj

    2016-01-01

    AIM To investigate expression of four alpha-carbonic anhydrases (CAs) in colorectal carcinomas (CRC) and compare the results with patients’ survival. METHODS Colorectal carcinoma samples from 539 CRC patients and control tissues were arranged as tissue microarrays and analyzed with antibodies against CA II, CA VII, CA IX, and CA XII. Intensity and extent of staining were both scored from 0 to 3 in each sample. These enzyme expression levels were then correlated to patients’ survival and clinicopathological parameters, which were tumor differentiation grade and stage, site of tumor, patients’ age, and gender. Kaplan-Meier analysis and Cox regression hazard ratio model were used to analyze survival data. RESULTS CA II and CA XII staining intensities correlated with patients’ survival in that higher expression indicated poorer prognosis. In Cox regression analysis one unit increase in the CA II intensity increased the hazard ratio to 1.19 fold (CI: 1.04-1.37, P = 0.009). A significant correlation was also found when comparing CA XII staining intensity with survival of CRC patients (HR = 1.18, 95%CI: 1.01-1.38, P = 0.036). The extent of CA XII immunostaining did not correlate to the patients’ survival (P = 0.242, Kaplan-Meier analysis). A significant interaction between age group and extent of the CA II staining was found. Increased extent of CA II had a significant hazard ratio among patients 65 years and older (1.42, 95%CI: 1.16-1.73, P = 0.0006). No correlations were found between CA VII (intensity P = 0.566, extent P = 0.495, Kaplan-Meier analysis), or CA IX (intensity P = 0.879, extent P = 0.315, Kaplan-Meier analysis) immunostaining results and survival, or the other parameters. CONCLUSION The present findings indicate that CA II and CA XII could be useful in predicting survival in CRC. PMID:27688658

  9. Carbonic anhydrase IX correlates with survival and is a potential therapeutic target for neuroblastoma.

    PubMed

    Ameis, Helen M; Drenckhan, Astrid; Freytag, Morton; Izbicki, Jakob R; Supuran, Claudiu T; Reinshagen, Konrad; Holland-Cunz, Stefan; Gros, Stephanie J

    2016-01-01

    Carbonic anhydrase IX (CAIX) is involved in pathological processes including tumorgenicity, metastases and poor survival in solid tumors. Twenty-two neuroblastoma samples of patients who were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated immunohistochemically for expression of CAIX. Results were correlated with clinical parameters and outcome. Neuroblastoma Kelly and SH-EP-Tet-21/N cells were examined for CAIX expression and inhibited with specific inhibitors, FC5-207A and FC8-325A. 32% of neuroblastoma tumors expressed CAIX. This was significantly associated with poorer survival. Kelly and SH-EP-Tet-21/N cells showed a major increase of CAIX RNA under hypoxic conditions. Proliferation of Kelly cells was significantly decreased by CAIX inhibitors, FC5-207A and FC8-325A, while proliferation of SH-EP-Tet-21/N cells was only significantly affected by FC8-325A. CAIX is a potent biomarker that predicts survival in neuroblastoma patients. CAIX-targeted therapy in neuroblastoma cell lines is highly effective and strengthens the potential of CAIX as a clinical therapeutic target in a selected patient collective.

  10. Imaging of carbonic anhydrase IX with an 111In-labeled dual-motif inhibitor

    PubMed Central

    Rowe, Steven P.; Banerjee, Sangeeta Ray; Gorin, Michael A.; Brummet, Mary; Lee, Hye Soo; Koo, Soo Min; Sysa-Shah, Polina; Mease, Ronnie C.; Nimmagadda, Sridhar; Allaf, Mohamad E.; Pomper, Martin G.

    2015-01-01

    We developed a new scaffold for radionuclide-based imaging and therapy of clear cell renal cell carcinoma (ccRCC) targeting carbonic anhydrase IX (CAIX). Compound XYIMSR-01, a DOTA-conjugated, bivalent, low-molecular-weight ligand, has two moieties that target two separate sites on CAIX, imparting high affinity. We synthesized [111In]XYIMSR-01 in 73.8–75.8% (n = 3) yield with specific radioactivities ranging from 118 – 1,021 GBq/μmol (3,200–27,600 Ci/mmol). Single photon emission computed tomography of [111In]XYIMSR-01 in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors revealed radiotracer uptake in tumor as early as 1 h post-injection. Biodistribution studies demonstrated 26% injected dose per gram of radioactivity within tumor at 1 h. Tumor-to-blood, muscle and kidney ratios were 178.1 ± 145.4, 68.4 ± 29.0 and 1.7 ± 1.2, respectively, at 24 h post-injection. Retention of radioactivity was exclusively observed in tumors by 48 h, the latest time point evaluated. The dual targeting strategy to engage CAIX enabled specific detection of ccRCC in this xenograft model, with pharmacokinetics surpassing those of previously described radionuclide-based probes against CAIX. PMID:26418876

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

    PubMed

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

    2016-12-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

  12. DISCLOSING THE INTERACTION OF CARBONIC ANHYDRASE IX WITH CULLIN-ASSOCIATED NEDD8-DISSOCIATED PROTEIN 1 BY MOLECULAR MODELING AND INTEGRATED BINDING MEASUREMENTS.

    PubMed

    Buonanno, Martina; Langella, Emma; Zambrano, Nicola; Succoio, Mariangela; Sasso, Emanuele; Alterio, Vincenzo; Di Fiore, Anna; Sandomenico, Annamaria; Supuran, Claudiu T; Scaloni, Andrea; Monti, Simona Maria; De Simone, Giuseppina

    2017-04-07

    Human Carbonic Anhydrase (hCA) IX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for several human cancers. In a recent paper we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CAND1 expression via shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible of the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking and site-directed mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX, these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and metastatic spread of tumors.

  13. Alternative splicing variants of carbonic anhydrase IX in human non-small cell lung cancer.

    PubMed

    Malentacchi, Francesca; Simi, Lisa; Nannelli, Caterina; Andreani, Matteo; Janni, Alberto; Pastorekova, Silvia; Orlando, Claudio

    2009-06-01

    In human cancers, carbonic anhydrase IX (CAIX) contributes to maintain intracellular and extracellular pH under hypoxic conditions, but also influences regulation of cell proliferation and tumor progression. CaIX was previously indicated as an independent prognostic marker in non-small cell lung carcinoma (NSCLC). Very recently a CAIX alternative splicing isoform, generating a transcript lacking of exons 8-9, was detected in cancer cells independently from the levels of hypoxia. This alternative splicing (AS) generates a truncated protein lacking the transmembrane region, the intracellular tail and the C-terminal of the catalytic domain and competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study we measured the mRNA expression of FL and AS CAIX isoforms in 101 NSCLC and in paired not affected tissues. The two isoforms were coexpressed in all NSCLC and normal tissues but while AS mRNA was prevalent in normal tissues (66+/-3%), the FL isoform was higher in NSCLC (58+/-2%, p=0.001). FL mRNA, but not AS, was statistically increased in NSCLC (p=0.01) and showed a statistical association with lymphnode involvement (p=0.009) and tumor stage (p=0.04). Global survival analysis of cancer/related death showed that high levels of FL mRNA were predictive of unfavorable outcome (p<0.0001) and shorter disease-free survival (p<0.0001). Multivariate analysis indicated that FL is an independent prognostic factor for overall survival and higher levels of mRNA in NSCLC sensibly increase hazard ratio ( approximately sixfold). In conclusion, our results seems to indicate that, at least in NSCLC, FL CAIX is the most accurate surrogate of hypoxic stress and represents the only variant with a prognostic role. These data indicate the importance of a separate measurement of the two isoforms in cancer and the need of an accurate re-evaluation of most studies on the clinical role of CAIX in cancer diagnosis.

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

    PubMed

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

    2015-10-20

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

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

    SciTech Connect

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

    2015-09-23

    Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2to HCO3$-$, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, anRcryst of 18.0% and anRfree of 21.2%. Finally, the binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.

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

    PubMed

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

    2009-02-17

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

  17. Carbonic anhydrase inhibitors: guaiacol and catechol derivatives effectively inhibit certain human carbonic anhydrase isoenzymes (hCA I, II, IX and XII).

    PubMed

    Scozzafava, Andrea; Passaponti, Maurizio; Supuran, Claudiu T; Gülçin, İlhami

    2015-01-01

    Carbonic anhydrases (CAs) are widespread metalloenzymes in higher vertebrates including humans. A series of phenolic compounds, including guaiacol, 4-methylguaiacol, 4-propylguaiacol, eugenol, isoeugenol, vanillin, syringaldehyde, catechol, 3-methyl catechol, 4-methyl catechol and 3-methoxy catechol were investigated for their inhibition of all the catalytically active mammalian isozymes of the Zn(2+)-containing CA (EC 4.2.1.1). All the phenolic compounds effectively inhibited human carbonic anhydrase isoenzymes (hCA I, II, IX and XII), with Kis in the range of 2.20-515.98 μM. The various isozymes showed diverse inhibition profiles. Among the tested phenolic derivatives, compounds 4-methyl catechol and 3-methoxy catechol showed potent activity as inhibitors of the tumour-associated transmembrane isoforms (hCA IX and XII) in the submicromolar range, with high selectivity. The results obtained from this research may lead to the design of more effective carbonic anhydrase isoenzyme inhibitors (CAIs) based on such phenolic compound scaffolds.

  18. Blood Oxygenation Level-dependent Magnetic Resonance Imaging of Breast Cancer: Correlation with Carbonic Anhydrase IX and Vascular Endothelial Growth Factor

    PubMed Central

    Wang, Ying; Liu, Min; Jin, Mu-Lan

    2017-01-01

    Background: Blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) is a functional MRI technique which involves using the paramagnetic properties of deoxyhemoglobin to image the local tissue oxygen concentration. The purpose of this study was to investigate whether BOLD-MRI could evaluate hypoxia and angiogenesis of breast invasive ductal carcinoma (IDC). Methods: Ninety-eight female patients with IDC were retrospectively included in this research. All patients underwent breast BOLD-MRI at 3.0 T before surgery. R2* values of BOLD-MRI were measured. The expression of carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF) was analyzed by immunohistochemistry. Spearman's correlation analysis was used to correlate R2* value with CA IX and VEGF levels. Results: Heterogeneous intensity on BOLD-MRI images was the main finding of IDCs. The mean R2* value was 52.8 ± 18.6 Hz. The R2* values in patients with axillary lymph node metastasis were significantly higher than the R2* values in patients without axillary lymph node metastasis (t = 2.882, P = 0.005). R2* values increased with CA IX level and positively correlated with the level of CA IX (r = 0.616, P < 0.001); however, R2* value had no significantly correlation with the level of VEGF (r = 0.110, P = 0.281). Conclusion: BOLD-MRI could noninvasively evaluate chronic hypoxia of IDC, but not angiogenesis. PMID:28051026

  19. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

    PubMed Central

    Ahlskog, J K J; Schliemann, C; Mårlind, J; Qureshi, U; Ammar, A; Pedley, R B; Neri, D

    2009-01-01

    Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications. PMID:19623173

  20. A highly selective space-folded photo-induced electron transfer fluorescent probe for carbonic anhydrase isozymes IX and its applications for biological imaging.

    PubMed

    Zhang, Shenyi; Yang, Chunmei; Lu, Weiqiang; Huang, Jin; Zhu, Weiping; Li, Honglin; Xu, Yufang; Qian, Xuhong

    2011-08-07

    The first highly selective and sensitive fluorescent probe Z1 for detection of carbonic anhydrase IX (CA IX) over isoforms CA I and CA II was developed. As demonstrated, Z1 worked effectively in both enzymatic systems and living hypoxia cells.

  1. Carbonic anhydrase IX (CA-IX) and human papillomavirus (HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study

    PubMed Central

    Liao, Shu-Yuan; Rodgers, William H.; Kauderer, James; Bonfiglio, Thomas A.; Walker, Joan L.; Darcy, Kathleen M.; Carter, Randy; Hatae, Masayuji; Levine, Lyuba; Spirtos, Nick M; Stanbridge, Eric J.

    2009-01-01

    High-risk human papillomavirus (H-HPV) infection is strongly linked to cervical neoplasia but its role in detecting glandular lesions is unclear. In the cervix, carbonic anhydrase IX (CA-IX) is expressed in cervical neoplasia, but rarely in the benign cervix. The diagnostic utility of these biomarkers was evaluated in women with a cytologic diagnosis of atypical glandular cells (AGC). H-HPV was detected using Hybrid Capture 2 (HC2) in liquid based cytology and CA-IX immunoreactivity was studied on conventional Pap smears. Of 403 patients, 111 (28%) were positive for significant cervical lesions (SCLs) including CIN2, CIN3, adenocarcinoma in situ or invasive carcinoma. CA-IX testing alone (n=403) had a sensitivity of 75%, 95%, or 65% for SCLs, significant glandular lesions (GLs) or squamous lesions (SLs), respectively, with a specificity of 88%, and a false negative rate (FNR defined as one minus negative predictive value) of 10%. Testing for H-HPV (n=122) had a sensitivity of 97%, 100%, or 96% for SCLs, GLs or SLs, respectively, with a specificity of 87%, and a FNRof 1%. The combination of CA-IX and H-HPV testing (n=122), collectively, had the same sensitivity, specificity and FNR for SCLs, GLs or SLs as H-HPV testing alone. The conclusions of this study are that both H-HPV and CA-IX testing are useful diagnostic markers for GLs. However, H-HPV testing is a better diagnostic marker for SLs. The combination of CA-IX with H-HPV testing does not improve the diagnostic accuracy for cervical neoplasia in women with AGC diagnosis over that of H-HPV testing alone. PMID:19670419

  2. Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors

    PubMed Central

    Faes, Seraina; Planche, Anne; Uldry, Emilie; Santoro, Tania; Pythoud, Catherine; Stehle, Jean-Christophe; Horlbeck, Janine; Letovanec, Igor; Riggi, Nicolo; Datta, Dipak; Demartines, Nicolas; Dormond, Olivier

    2016-01-01

    The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation. PMID:27153561

  3. Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I, II and IX with some hydroxylic compounds.

    PubMed

    Durdagi, Serdar; Korkmaz, Neslihan; Işık, Semra; Vullo, Daniela; Astley, Demet; Ekinci, Deniz; Salmas, Ramin E; Senturk, Murat; Supuran, Claudiu T

    2016-12-01

    A series of hydroxylic compounds (1-10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068-4003, 0.012-9.9 and 0.025-115 μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the KI values were calculated.

  4. Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates.

    PubMed

    Innocenti, Alessio; Vullo, Daniela; Scozzafava, Andrea; Casey, Joseph R; Supuran, Claudiut

    2005-02-01

    A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate (K(A) around 0.1 microM), whereas formate, malonate, malate, and benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K(I) of 99 nM for citrate and oxalate, of 2.8 microM for malonate and of 14.5 microM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K(I) of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K(I) of 5.5mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (K(I) in the range of 1.12-7.42 mM), except acetate, lactate, and benzoate, which showed a K(I)>150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to

  5. Evaluation of selenide, diselenide and selenoheterocycle derivatives as carbonic anhydrase I, II, IV, VII and IX inhibitors.

    PubMed

    Angeli, Andrea; Tanini, Damiano; Viglianisi, Caterina; Panzella, Lucia; Capperucci, Antonella; Menichetti, Stefano; Supuran, Claudiu T

    2017-04-15

    A series of selenides, diselenides and organoselenoheterocycles were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV, VII and IX, involved in a variety of diseases among which glaucoma, retinitis pigmentosa, epilepsy, arthritis and tumors etc. These investigated compounds showed inhibitory action against these isoforms and some of them were selective for inhibiting the cytosolic over the membrane-bound isoforms, thus making them interesting leads for the development of isoform-selective inhibitors.

  6. Synthesis and biological evaluation of cyclic imides incorporating benzenesulfonamide moieties as carbonic anhydrase I, II, IV and IX inhibitors.

    PubMed

    Abdel-Aziz, Alaa A-M; Angeli, Andrea; El-Azab, Adel S; Abu El-Enin, Mohamed A; Supuran, Claudiu T

    2017-03-01

    A group of cyclic imides was synthesized by reaction of amino-substituted benzenesulfonamides with a series of acid anhydrides such as succinic, maleic, tetrahydrophthalic, pyrazine-2,3-dicarboxylic acid anhydride, and substituted phthalic anhydrides. The synthesized sulfonamides were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV and IX, involved in a variety of diseases among which glaucoma, retinitis pigmentosa, etc. Some of these sulfonamides showed effective inhibitory action (in the nanomolar range) against the cytosolic isoform hCA II and the transmembrane, tumor-associated one hCA IX, making them interesting candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes are involved. hCA I and IV were on the other hand less inhibited by these sulfonamides, with inhibition constants in the micromolar range.

  7. 5-Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic anhydrases IX and XII.

    PubMed

    Cvijetić, Ilija N; Tanç, Muhammet; Juranić, Ivan O; Verbić, Tatjana Ž; Supuran, Claudiu T; Drakulić, Branko J

    2015-08-01

    Inhibitory activity of a congeneric set of 23 phenyl-substituted 5-phenyl-pyrazole-3-carboxylic acids toward human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II, IX and XII was evaluated by a stopped-flow CO2 hydrase assay. These compounds exerted a clear, selective inhibition of hCA IX and XII over hCAI and II, with Ki in two to one digit micromolar concentrations (4-50 μM). Derivatives bearing bulkier substituents in para-position of the phenyl ring inhibited hCA XII at one-digit micromolar concentrations, while derivatives having alkyl substituents in both ortho- and meta-positions inhibited hCA IX with Kis ranging between 5 and 25 μM. Results of docking experiments offered a rational explanation on the selectivity of these compounds toward CA IX and XII, as well as on the substitution patterns leading to best CA IX or CA XII inhibitors. By examining the active sites of these four isoforms with GRID generated molecular-interaction fields, striking differences between hCA XII and the other three isoforms were observed. The field of hydrophobic probe (DRY) appeared significantly different in CA XII active site, comparing to other three isoforms studied. To the best of our knowledge such an observation was not reported in literature so far. Considering the selectivity of these carboxylates towards membrane-associated over cytosolic CA isoforms, the title compounds could be useful for the development of isoform-specific non-sulfonamide CA inhibitors.

  8. Carbonic anhydrase IX induction defines a heterogeneous cancer cell response to hypoxia and mediates stem cell-like properties and sensitivity to HDAC inhibition

    PubMed Central

    Wigfield, Simon; Buffa, Francesca; McGowan, Simon; Baban, Dilair; Li, Ji-liang; Harris, Adrian L.

    2015-01-01

    Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. Here, we report that hypoxia promotes tumour heterogeneity through the epigenetic regulation of CAIX. Based on hypoxic CAIX expression we identify and characterize two distinct populations of tumour cells, one that has inducible expression of CAIX and one that does not. The CAIX+ve population is enriched with cells expressing cancer stem cell markers and which have high self-renewal capacity. We show that differential CAIX expression is due to differences in chromatin structure. To further investigate the relationship between chromatin organization and hypoxic induction of CAIX expression we investigated the effect of JQ1 an inhibitor of BET bromodomain proteins and A366 a selective inhibitor of the H3K9 methyltransferase G9a/GLP. We identified that these drugs were able to modulate hypoxic CAIX expression induction. This further highlights the role of epigenetic modification in adaption to hypoxia and also in regulation of heterogeneity of cells within tumours. Interestingly, we identified that the two subpopulations show a differential sensitivity to HDAC inhibitors, NaBu or SAHA, with the CAIX positive showing greater sensitivity to treatment. We propose that drugs modulating chromatin regulation of expression may be used to reduce heterogeneity induced by hypoxia and could in combination have significant clinical consequences. PMID:26305601

  9. Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer

    PubMed Central

    Ivanov, Sergey; Liao, Shu-Yuan; Ivanova, Alla; Danilkovitch-Miagkova, Alla; Tarasova, Nadezhda; Weirich, Gregor; Merrill, Marsha J.; Proescholdt, Martin A.; Oldfield, Edward H.; Lee, Joshua; Zavada, Jan; Waheed, Abdul; Sly, William; Lerman, Michael I.; Stanbridge, Eric J.

    2001-01-01

    An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors. PMID:11238039

  10. Carbonic anhydrase IX promotes myeloid-derived suppressor cell mobilization and establishment of a metastatic niche by stimulating G-CSF production.

    PubMed

    Chafe, Shawn C; Lou, Yuanmei; Sceneay, Jaclyn; Vallejo, Marylou; Hamilton, Melisa J; McDonald, Paul C; Bennewith, Kevin L; Möller, Andreas; Dedhar, Shoukat

    2015-03-15

    The mobilization of bone marrow-derived cells (BMDC) to distant tissues before the arrival of disseminated tumor cells has been shown preclinically to facilitate metastasis through the establishment of metastatic niches. Primary tumor hypoxia has been demonstrated to play a pivotal role in the production of chemokines and cytokines responsible for the mobilization of these BMDCs, especially in breast cancer. Carbonic anhydrase IX (CAIX, CA9) expression is highly upregulated in hypoxic breast cancer cells through the action of hypoxia-inducible factor-1 (HIF1). Preclinical evidence has demonstrated that CAIX is required for breast tumor growth and metastasis; however, the mechanism by which CAIX exerts its prometastatic function is not well understood. Here, we show that CAIX is indispensable for the production of granulocyte colony-stimulating factor (G-CSF) by hypoxic breast cancer cells and tumors in an orthotopic model. Furthermore, we demonstrate that tumor-expressed CAIX is required for the G-CSF-driven mobilization of granulocytic myeloid-derived suppressor cells (MDSC) to the breast cancer lung metastatic niche. We also determined that CAIX expression is required for the activation of NF-κB in hypoxic breast cancer cells and constitutive activation of the NF-κB pathway in CAIX-depleted cells restored G-CSF secretion. Together, these findings identify a novel hypoxia-induced CAIX-NF-κB-G-CSF cellular signaling axis culminating in the mobilization of granulocytic MDSCs to the breast cancer lung metastatic niche.

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

    PubMed

    Kamstra, Rhiannon L; Floriano, Wely B

    2014-11-01

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

  12. Carbonic anhydrase IX and human papillomavirus as diagnostic biomarkers of cervical dysplasia/neoplasia in women with a cytologic diagnosis of atypical glandular cells: a Gynecologic Oncology Group study in United States.

    PubMed

    Liao, Shu-Yuan; Rodgers, William H; Kauderer, James; Bonfiglio, Thomas A; Walker, Joan L; Darcy, Kathleen M; Carter, Randy; Hatae, Masayuji; Levine, Lyuba; Spirtos, Nick M; Stanbridge, Eric J

    2009-11-15

    High-risk human papillomavirus (H-HPV) infection is strongly linked to cervical neoplasia, but its role in detecting glandular lesions (GLs) is unclear. In the cervix, carbonic anhydrase IX (CA-IX) is expressed in cervical neoplasia, but rarely in the benign cervix. The diagnostic utility of these biomarkers was evaluated in women with a cytologic diagnosis of atypical glandular cells (AGC). H-HPV was detected using hybrid capture 2 (HC2) in liquid-based cytology, and CA-IX immunoreactivity was studied on conventional Pap smears. Of 403 patients, 111 (28%) were positive for significant cervical lesions (SCLs) including CIN2, CIN3, adenocarcinoma in situ or invasive carcinoma. CA-IX testing alone (n = 403) had a sensitivity of 75, 95 or 65% for SCLs, significant GLs or squamous lesions (SLs), respectively, with a specificity of 88% and a false negative rate (FNR defined as 1 minus negative predictive value) of 10%. Testing for H-HPV (n = 122) had a sensitivity of 97, 100 or 96% for SCLs, GLs or SLs, respectively, with a specificity of 87% and a FNR of 1%. The combination of CA-IX and H-HPV testing (n = 122), collectively, had the same sensitivity, specificity and FNR for SCLs, GLs or SLs as H-HPV testing alone. The conclusions of our study are that both H-HPV and CA-IX testing are useful diagnostic markers for GLs. However, H-HPV testing is a better diagnostic marker for SLs. The combination of CA-IX with H-HPV testing does not improve the diagnostic accuracy for cervical neoplasia in women with AGC diagnosis over that of H-HPV testing alone.

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

    PubMed

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

    2014-01-01

    A new approach in the treatment of clear cell renal carcinoma (ccRCC) is radioimmunotherapy (RIT) using anti-carbonic anhydrase IX (CAIX) antibody G250. To investigate the potential of RIT with lutetium 177 (177Lu)-labeled G250, we conducted a protein dose escalation study and subsequently an RIT study in mice with intraperitoneally growing ccRCC lesions. Mice with intraperitoneal xenografts were injected with 1, 3, 10, 30, or 100 μg of G250 labeled with 10 MBq indium 111 (111In) to determine the optimal protein dose. The optimal protein dose determined with imaging and biodistribution studies was used in a subsequent RIT experiment in three groups of 10 mice with intraperitoneal SK-RC-52 tumors. One group received 13 MBq 177Lu-DOTA-G250, a control group received 13 MBq nonspecific 177Lu-MOPC21, and the second control group was not treated and received 20 MBq 111In-DOTA-G250. The optimal G250 protein dose to target ccRCC in this model was 10 μg G250. Treatment with 13 MBq 177Lu-DOTA-G250 was well tolerated and resulted in significantly prolonged median survival (139 days) compared to controls (49-53 days, p  =  .015), indicating that RIT has potential in this metastatic ccRCC model.

  14. DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

    PubMed Central

    Kaluzová, Milota; Kaluz, Stefan; Lerman, Michael I.; Stanbridge, Eric J.

    2004-01-01

    We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53+/+ but not the isogenic p53−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented. PMID:15199132

  15. Carbonic anhydrase IX (CA-IX) and high-risk human papillomavirus (H-HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in Japanese women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study

    PubMed Central

    Liao, S-Y; Rodgers, W H; Kauderer, J; Bonfiglio, T A; Darcy, K M; Carter, R; Levine, L; Spirtos, N M; Susumu, N; Fujiwara, K; Walker, J L; Hatae, M; Stanbridge, E J

    2011-01-01

    Background: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. Methods: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. Results: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). Conclusion: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV. PMID:21157448

  16. A survey of carbonic anhydrase mRNA expression in enamel cells

    PubMed Central

    LACRUZ, Rodrigo S.; HILVO, Mika; KURTZ, Ira; PAINE, Michael L.

    2010-01-01

    Enamel formation requires rigid control of pH homeostasis during all stages of development to prevent disruptions to crystal growth. The acceleration of the generation of bicarbonate by carbonic anhydrases (CA) has been suggested as one of the pathways used by ameloblasts cells to regulate extracellular pH yet only two isozymes (CA II and CA VI) have been reported to date during enamel formation. The mammalian CA family contains 16 different isoforms of which 13 are enzymatically active. We have conducted a systematic screening by RT-PCR on the expression of all known CA isoforms in mouse enamel organ epithelium (EOE) cells dissected from new born, in secretory ameloblasts derived from 7-day old animals, and in the LS8 ameloblast cell line. Results show that all CA isoforms are expressed by EOE/ameloblast cells in vivo. The most highly expressed are the catalytic isozymes CA II, VI, IX, and XIII, and the acatalytic CA XI isoform. Only minor differences were found in CA expression levels between 1-day EOE cells and 7-day old secretory stage ameloblasts, whereas LS8 cells expressed fewer CA isoforms than both of these. The broad expression of CAs by ameloblasts reported here contributes to our understanding of pH homeostasis during enamel development and demonstrates its complexity. Our results also highlight the critical role that regulation of pH plays during the development of enamel. PMID:20175995

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

    PubMed

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

    2014-11-01

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

  18. Comparison of expression systems for human fucosyltransferase IX.

    PubMed

    Stacke, Christina; Ziegelmüller, Patrick; Hahn, Ulrich

    2010-01-01

    Human fucosyltransferase IX (hFucT-IX) is a highly conserved alpha1,3 fucosyltransferase with a distinct acceptor and site specificity. hFucT-IX catalyses the transfer of activated fucose to a sugar acceptor, thereby forming the Lewis x epitope. This epitope is responsible for recognition phenomena throughout the body e.g. in tumour growth. Detailed characterisation of hFucT-IX structure-function relationships by kinetic and X-ray structure analysis is prerequisite to the development of enzyme inhibitors for clinical applications such as the suppression of tumour metastasis. For these analyses substantial amounts of hFucT-IX are desirable. Since hFucT-IX is not present in considerable amounts in common cells an overproduction of recombinant hFucT-IX is appropriate. To evaluate the best system for this overproduction we compared different strategies employing prokaryotes (Escherichia coli), mammalian cells and insect cells. Insect cells were tested using stable and baculoviral expression strategies. Current results favour the use of the baculoviral expression system for further experiments.

  19. Non-invasive Detection of Breast Cancer Lymph Node Metastasis using Carbonic Anhydrases IX and XII Targeted Imaging Probes

    PubMed Central

    Tafreshi, Narges K.; Bui, Marilyn M.; Bishop, Kellsey; Lloyd, Mark C.; Enkemann, Steven A.; Lopez, Alexis S.; Abrahams, Dominique; Carter, Bradford W.; Vagner, Josef; Grobmyer, Stephen R.; Gillies, Robert J.; Morse, David L.

    2014-01-01

    Purpose To develop targeted molecular imaging probes for the non-invasive detection of breast cancer lymph node metastasis. Methods Six cell surface or secreted markers were identified by expression profiling and from the literature as being highly expressed in breast cancer lymph node metastases. Two of these markers were cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) and were validated for protein expression by immunohistochemistry (IHC) of patient tissue samples on a breast cancer tissue microarray containing 47 normal breast tissue samples, 42 ductal carcinoma in situ, 43 invasive ductal carcinomas without metastasis, 46 invasive ductal carcinomas with metastasis and 49 lymph node macrometastases of breast carcinoma. Targeted probes were developed by conjugation of CAIX and CAXII specific monoclonal antibodies (mAbs) to a near-infrared fluorescent dye. Results Together, these two markers were expressed in 100% of the lymph node metastases surveyed. Selectivity of the imaging probes were confirmed by intravenous injection into nude mice bearing mammary fat pad tumors of marker expressing cells, and non-expressing cells or by pre-injection of unlabeled antibody. Imaging of LN metastases showed that peritumorally-injected probes detected nodes harboring metastatic tumor cells. As few as 1,000 cells were detected, as determined by implanting, under ultrasound guidance, a range in number of CAIX and CAXII expressing cells into the axillary LNs. Conclusion These imaging probes have potential for non-invasive staging of breast cancer in the clinic and elimination of unneeded surgery, which is costly and associated with morbidities. PMID:22016510

  20. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

  1. Open saccharin-based secondary sulfonamides as potent and selective inhibitors of cancer-related carbonic anhydrase IX and XII isoforms.

    PubMed

    D'Ascenzio, Melissa; Guglielmi, Paolo; Carradori, Simone; Secci, Daniela; Florio, Rosalba; Mollica, Adriano; Ceruso, Mariangela; Akdemir, Atilla; Sobolev, Anatoly P; Supuran, Claudiu T

    2017-12-01

    A large number of novel secondary sulfonamides based on the open saccharin scaffold were synthesized and evaluated as selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). They were obtained by reductive ring opening of the newly synthesized N-alkylated saccharin derivatives and were shown to be inactive against the two cytosolic off-target hCA I and II (Kis > 10 µM). Interestingly, these compounds inhibited hCA IX in the low nanomolar range with Kis ranging between 20 and 298 nM and were extremely potent inhibitors of hCA XII isoenzyme (Kis ranging between 4.3 and 432 nM). Since hCA IX and XII are the cancer-related isoforms recently validated as drug targets, these results represent an important goal in the development of new anticancer candidates. Finally, a computational approach has been performed to better correlate the biological data to the binding mode of these inhibitors.

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

    PubMed

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

    2015-08-01

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

  3. Constitutive episomal expression of polypeptide IX (pIX) in a 293-based cell line complements the deficiency of pIX mutant adenovirus type 5.

    PubMed Central

    Caravokyri, C; Leppard, K N

    1995-01-01

    The human adenovirus type 5 capsid is composed of a number of distinct polypeptides. It has been shown previously that one of these, polypeptide IX (pIX), is not absolutely required for the production of viable virus. However, viruses lacking this polypeptide have a significantly reduced packaging limit and, in the one case studied, also show a thermolabile virion phenotype. This report describes the use of eukaryotic episomal vectors based on the Epstein-Barr virus replicon to generate cells which stably express pIX. These cells provide pIX that is efficiently incorporated into virions that are genetically pIX-; such enhanced thermostability. These cells have also been used to isolate a genetically pIX- virus having a genome of length some 2.3 kbp in excess of the previously defined packaging limit for pIX- virus; the resulting virions have wild-type thermostability. These cells expand the theoretical capacity of adenovirus vectors for foreign DNA to around 9.2 kbp and may therefore be useful in gene therapy applications in which vector capacity is limiting. PMID:7474071

  4. Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with phosphates, carbamoyl phosphate, and the phosphonate antiviral drug foscarnet.

    PubMed

    Rusconi, Stefano; Innocenti, Alessio; Vullo, Daniela; Mastrolorenzo, Antonio; Scozzafava, Andrea; Supuran, Claudiu T

    2004-12-06

    A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.

  5. Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate.

    PubMed

    Innocenti, Alessio; Vullo, Daniela; Scozzafava, Andrea; Supuran, Claudiu T

    2005-02-01

    The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8

  6. Near-Infrared Fluorescence Imaging of Carbonic Anhydrase IX in Athymic Mice Bearing HT-29 Tumor Xenografts

    PubMed Central

    2016-01-01

    Near-infrared fluorescence (NIRF) imaging technology is a highly sensitive imaging modality and has been widely used in noninvasively studying the status of receptor expression in small animal models, with an appropriate NIRF probe targeting a specific receptor. In this report, Cy5.5-conjugated anti-CAIX monoclonal antibody (Mab-Cy5.5) was evaluated in athymic mice bearing HT-29 tumor xenografts in order to investigate the effect of conjugate on tumor targeting efficacy. In vitro binding studies showed that Mab-Cy5.5 could specifically bind to the cells which expressed CAIX. Results from in vivo imaging showed that HT-29 tumor xenografts can be clearly visualized at 48 h after injection of Mab-Cy5.5, and in the blocking experiment, free anti-CAIX antibody effectively blocked the concentration of Mab-Cy5.5 in the tumors. Western blotting and immunohistochemistry analysis of HT-29 tumor xenografts verified the expression of CAIX in HT-29 tumors. Mab-Cy5.5 could specifically bind to the tumors which expressed CAIX. These results suggested that Mab-Cy5.5 was suitable for CAIX expression imaging in the preclinical research. PMID:27652266

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

    PubMed

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

    2008-09-01

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

  8. Synthesis of bulky-tailed sulfonamides incorporating pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl) moieties and evaluation of their carbonic anhydrases I, II, IV and IX inhibitory effects.

    PubMed

    Fares, Mohamed; Eladwy, Radwa A; Nocentini, Alessio; El Hadi, Soha R Abd; Ghabbour, Hazem A; Abdel-Megeed, Ashraf; Eldehna, Wagdy M; Abdel-Aziz, Hatem A; Supuran, Claudiu T

    2017-04-01

    Using celecoxib as lead, two novel series of sulfonamides incorporating the pyridotriazolopyrimidine scaffold have been synthesized and evaluated in vitro as inhibitors against four relevant human (h) carbonic anhydrases (CAs, EC 4.2.1.1), the cytosolic and ubiquitous hCA I and II as well as the transmembrane hCA IV and hCA IX. Most of the reported sulfonamides acted as efficient, low micromolar inhibitors of hCAI, II and IV, whereas they displayed higher efficacy in inhibiting the tumor-associated isoform hCA IX. Many derivates herein reported showed better hCA IX versus hCA II selectivity ratios compared to celecoxib or acetazolamide. Considering isoform IX is a validated target for the diagnosis and treatment of hypoxic tumors, discovery of selective CA IX inhibitors represents a promising step to unveil more effective anticancer therapies.

  9. Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

    PubMed Central

    Liu, Xiaochen; Lu, Deyi; Bowser, Robert; Liu, Jian

    2016-01-01

    Carbonic anhydrase I (CA1) is the cytosolic isoform of mammalian α-CA family members which are responsible for maintaining pH homeostasis in the physiology and pathology of organisms. A subset of CA isoforms are known to be expressed and function in the central nervous system (CNS). CA1 has not been extensively characterized in the CNS. In this study, we demonstrate that CA1 is expressed in the motor neurons in human spinal cord. Unexpectedly, a subpopulation of CA1 appears to be associated with endoplasmic reticulum (ER) membranes. In addition, the membrane-associated CA1s are preferentially upregulated in amyotrophic lateral sclerosis (ALS) and exhibit altered distribution in motor neurons. Furthermore, long-term expression of CA1 in mammalian cells activates apoptosis. Our results suggest a previously unknown role for CA1 function in the CNS and its potential involvement in motor neuron degeneration in ALS. PMID:27809276

  10. Erythroid expression and DNAaseI-hypersensitive sites of the carbonic anhydrase 1 gene.

    PubMed Central

    Sowden, J; Edwards, M; Morrison, K; Butterworth, P H; Edwards, Y H

    1992-01-01

    The carbonic anhydrase 1 gene is expressed in adult human and mouse erythroid cells and colon epithelia from two distinct promoters. We have explored the erythroid promoter for cis-acting sequences involved in transcription using DNAaseI as a probe. Two DNAaseI-hypersensitive sites (DHS-1 and DHS-2) have been identified in the distal erythroid promoter in CA1-expressing erythroleukaemic cells. These sites are present at low levels in K562 cells, which have a foetal/embryonic phenotype and do not express CA1. DHS-1 and DHS-2 are not present in non-erythroid cells, including colon cells, which express CA1 from the proximal colon promoter. DHS-1 and DHS-2 were also generated in an heterologous CA1 gene containing 5 kb of erythroid promoter sequence after transfection into erythroid cells, including K562 cells. These transfection studies showed that both this fragment, and an abbreviated 817 bp promoter fragment which contains only DHS-1, were sufficient to confer erythroid-specific expression to a reporter gene. These promoters were active in cell lines expressing CA1 and in K562 cells. This latter observation implies that a developmental repressor factor is both present in K562 cells and binds to a cis-acting sequence that is absent from the sequence 5 kb upstream of the erythroid transcription start site. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1463458

  11. Observed surface lysine acetylation of human carbonic anhydrase II expressed in Escherichia coli

    PubMed Central

    Mahon, Brian P; Lomelino, Carrie L; Salguero, Antonieta L; Driscoll, Jenna M; Pinard, Melissa A; McKenna, Robert

    2015-01-01

    Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems. PMID:26266677

  12. [Construction of nonsense-mutated eukaryotic expression vector of factor IX gene and its expression in COS-7 cells].

    PubMed

    Nie, Xin; Yang, Lin-Hua; Chai, Bao-Feng; Shen, Quan; Zhang, Yuan; Zhang, Yao-Fang; Chen, Jian-Fang

    2010-06-01

    The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fIX gene, using pcDNA3.1 plasmid containing fIX cDNA as template, and to identify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirmed by DNA sequencing. COS-7 cells were transfected with either the wild-type or mutated fIX expression constructs, then the relative expression levels of fIX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nonsense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fIX gene have been successfully constructed and can express in COS-7 cells, which provides the material basis for further researches on mechanism and treatment of FIX deficiency and the function defects caused by nonsense mutation.

  13. Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis.

    PubMed

    Kaunisto, K; Fleming, R E; Kneer, J; Sly, W S; Rajaniemi, H

    1999-12-01

    Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.

  14. Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development.

    PubMed

    Huang, Sherri; Holmes, Michael J; Radke, Joshua B; Hong, Dong-Pyo; Liu, Ting-Kai; White, Michael W; Sullivan, William J

    2017-01-01

    Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and

  15. Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

    PubMed Central

    Huang, Sherri; Holmes, Michael J.; Radke, Joshua B.; Hong, Dong-Pyo; Liu, Ting-Kai; White, Michael W.

    2017-01-01

    ABSTRACT Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction—a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in

  16. Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO2-Requiring Phenotype 1

    PubMed Central

    Price, G. D.; Badger, M. R.

    1989-01-01

    Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (Ci) and low Ci grown cells leads the creation of a high Ci requiring phenotype causing: (a) a dramatic increase in the K0.5 (Ci) for photosynthesis, (b) a loss of the ability to accumulate internal Ci, and (c) a decrease in the lag between the initial Ci accumulation following illumination and the efflux of CO2 from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO2 concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and Ci accumulation is developed in which the carboxysome plays a central role as both the site of CO2 generation from HCO3− and a resistance barrier to CO2 efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells. Images Figure 7 PMID:16667062

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-12-01

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

  19. Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm*

    PubMed Central

    Wandernoth, Petra M; Mannowetz, Nadja; Szczyrba, Jaroslaw; Grannemann, Laura; Wolf, Anne; Becker, Holger M.; Sly, William S.; Wennemuth, Gunther

    2015-01-01

    HCO3− is a key factor in the regulation of sperm motility. High concentrations of HCO3− in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3−, represent potential candidates in the regulation of the HCO3− homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3− homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3−-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3− or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3−. These results suggest that CAII and CAIV are required for optimal fertilization. PMID:26487715

  20. A combined theoretical and spectroscopic study of 4,6-di-O-acetyl-2,3-dideoxy-D-erythro-hex-2-enopyranosyl sulfamide: a novel glycosyl carbonic anhydrase IX inhibitor.

    PubMed

    Lavecchia, Martín J; Diez, Reinaldo Pis; Colinas, Pedro A

    2011-02-15

    The novel 4,6-di-O-acetyl-2,3-dideoxy-D-erythro-hex-2-enopyranosyl sulfamide, which exhibits selectivity for inhibiting isoform IX of carbonic anhydrase as overexpressed in many tumors, has been investigated from a combined theoretical and spectroscopic point of view. The conformational study of the compound shows that the α-anomeric form is more stable than the β-anomeric form from a thermodynamic point of view after including solvent effects. This fact suggests that the synthesis reaction could take place mainly under thermodynamic control as the main experimental product is the α-anomeric form of the sulfamide. Calculated α/β ratio is about 95:5, in excellent agreement with experimental data. Optimized geometries of the α-anomeric form agree quite well with crystallographic data. The inclusion of a solvent has negligible effects on the conformations. A detailed analysis of some geometric parameters shed light into the conformational behavior of the sulfamide in terms of both exo- and endo-anomeric effects and antiperiplanar relationships. Natural bond orbital calculations confirm those findings. Several intramolecular hydrogen bonds, characterized through the Atoms-in-Molecules theory, were found in the stable conformers. They, however, seem to play no relevant role in determining the relative stability of α conformers with respect to the β ones. Calculated (1)H and (13)C NMR chemical shifts support previous findings concerning configuration and conformation assignments of the title sulfamide. The IR spectrum of the compound is recorded and reported for the first time and the assignment of some of the most important bands is accomplished with the aid of calculated harmonic vibrational frequencies.

  1. Expression of human factor IX in rat capillary endothelial cells: Toward somatic gene therapy for hemophilia B

    SciTech Connect

    Shounan Yao; Wilson, J.M.; Nabel, E.G.; Kurachi, Sumiko; Hachiya, H.L.; Kurachi, Kotoku )

    1991-09-15

    In aiming to develop a gene therapy approach for hemophilia B, the authors expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including {gamma}-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

  2. The effects of CA IX catalysis products within tumor microenvironment.

    PubMed

    Santi, Alice; Caselli, Anna; Paoli, Paolo; Corti, Denise; Camici, Guido; Pieraccini, Giuseppe; Taddei, Maria Letizia; Serni, Sergio; Chiarugi, Paola; Cirri, Paolo

    2013-10-29

    Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.

  3. Comparison of protoporphyrin IX content and related gene expression in the tissues of chickens laying brown-shelled eggs.

    PubMed

    Li, Guangqi; Chen, Sirui; Duan, Zhongyi; Qu, Lujiang; Xu, Guiyun; Yang, Ning

    2013-12-01

    Protoporphyrin IX (PpIX), an immediate precursor of heme, is the main pigment resulting in the brown coloration of eggshell. The brownness and uniformity of the eggshell are important marketing considerations. In this study, 9 chickens laying darker brown shelled eggs and 9 chickens laying lighter brown shelled eggs were selected from 464 individually caged layers in a Rhode Island Red pureline. The PpIX contents were measured with a Microplate Reader at the wavelength of 412 nm and were compared in different tissues of the 2 groups. Although no significant difference in serum, bile, and excreta was found between the 2 groups, PpIX content in the shell gland and eggshell of the darker group was higher than in those of the lighter group, suggesting that PpIX was synthesized in the shell gland. We further determined the expression levels of 8 genes encoding enzymes involved in the heme synthesis and transport in the liver and shell gland at 6 h postoviposition by quantitative PCR. The results showed that expression of aminolevulinic acid synthase-1 (ALAS1) was higher in the liver of hens laying darker brown shelled eggs, whereas in the shell gland the expression levels of ALAS1, coproporphyrinogen oxidase (CPOX), ATP-binding cassette family members ABCB7 and ABCG2, and receptor for feline leukemia virus, subgroup C (FLVCR) were significantly higher in the hens laying darker brown shelled eggs. Our results demonstrated that hens laying darker brown shelled eggs could deposit more PpIX onto the eggshell and the brownness of the eggshell was dependent on the total quantity of PpIX in the eggshell. More heme was synthesized in the liver and shell gland of hens laying darker brown shelled eggs than those of hens laying lighter brown shelled eggs. High expression level of ABCG2 might facilitate the accumulation of PpIX in the shell gland.

  4. Expression pattern and intensity of protoporphyrin IX induced by liposomal 5-aminolevulinic acid in rat pilosebaceous unit throughout hair cycle.

    PubMed

    Han, Insook; Jun, Mee Sook; Kim, Soo-Kyun; Kim, Moonkyu; Kim, Jung Chul

    2005-11-01

    We have developed liposomal formulation of 5-aminolevulinic acid (ALA) to enhance topical delivery and examined ALA-induced protoporpyrin (PpIX) expression in rat pilosebaceous unit throughout hair cycle. Two types of liposomes--glycerol dilaulate (GDL) and phosphatidylcholine (PC)--were formulated and both liposomal ALA increased PpIX expression in rat dorsal skin and pilosebaceous units when compared with free ALA. However, iontophoresis combined with liposomal ALA reduced the expression intensity of PpIX in hair bulbs although it achieved deeper and wider expression of PpIX through transfollicular pathway. After topical application in intact or depilated rat skin, liposomal ALA produced excellent PpIX expression in pilosebaceous units. The expression pattern and intensity of PpIX changed in hair cycle-dependent manner: specific expression only in sebaceous glands was observed at telogen; strong expression in whole pilosebaceous units was shown at anagen with intense expressions in hair bulbs and sebaceous glands; and a pattern similar to anagen but reduced intensity in the hair bulbs was seen at catagen. Throughout hair cycle, the expression pattern and intensity were dramatically changed in hair follicular epithelial cells depending on the cell density and proliferation activity of those cells, whereas those were consistent in sebaceous glands regardless of hair cycle. Little expression was shown in dermis. Photoactivation effect of 20% liposomal ALA-PDT using a red filtered-halogen lamp damaged sebaceous glands, hair follicles and epidermal layers. Formation of a thicker epidermal layer was observed, and hair induction after depilation was inhibited along with damage in sebaceous glands.

  5. The use of CA-IX as a diagnostic method for oral leukoplakia.

    PubMed

    Pérez-Sayáns, M; Suárez-Peñaranda, J M; Torres-López, M; Supuran, C T; Gándara-Vila, P; Gayoso-Diz, P; Barros-Angueira, F; Gallas-Torreira, M; García-García, A

    2015-02-01

    The presence and degree of dysplasia are important diagnostic and prognostic criteria for oral leukoplakia, but evaluation of dysplasia is difficult and subjective. Carbonic anhydrase-IX (CA-IX) is expressed primarily in tumor cells and is considered a specific hypoxia marker. We investigated the role of CA-IX in oral leukoplakia. We investigated 30 specimens of oral leukoplakia and 35 dysplasia specimens adjacent to the tumor margin. We analyzed clinical variables including age, sex, degree of dysplasia, and smoking, clinical appearance of leukoplakia, number of lesions, location, size, clinical monitoring, malignant transformation and recurrence. For the immunohistochemical study, we used a noncommercial monoclonal antibody against human CA-IX MAb M75. We found greater CA-IX positivity in nonsmokers, erythroplakia and mottled leukoplakia, those located on the tongue, patients with multiple lesions, 2-4 cm leukoplakias and in recurrent cases, although differences were not statistically significant. All lesions in all samples without dysplasia were negative for CA-IX; however, for all other categories of dysplasia, the percentages of positivity and negativity varied. Regarding the diagnostic index values, we found a sensitivity of 32%, specificity of 100%, a positive predictive value of 100% and a negative predictive value of 13%. Leukoplakias appear mainly in females and potentially are malignant; more than 90% have some degree of dysplasia, and therefore require close clinical and histopathological monitoring. The CA-IX immunohistochemical marker may be useful for screening samples without dysplasia owing to its high specificity.

  6. Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo.

    PubMed

    Swiderski, R E; Solursh, M

    1992-05-01

    Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24-36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5' region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.

  7. Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum).

    PubMed

    Zhang, Hui; Zhao, Lingxia; Chen, Yuhui; Cui, Lijie; Ren, Weiwei; Tang, Kexuan

    2007-10-01

    In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

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

    PubMed

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

    2014-01-16

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

  9. Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1).

    PubMed

    Fisher, S Zoë; Tariku, Iyerus; Case, Nicolette M; Tu, Chingkuang; Seron, Teri; Silverman, David N; Linser, Paul J; McKenna, Robert

    2006-08-01

    Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.

  10. Induction of aminolevulinic acid synthase gene expression and enhancement of metabolite, protoporphyrin IX, excretion by organic germanium.

    PubMed

    Nakamura, Takashi; Saito, Miki; Shimada, Yasuhiro; Fukaya, Haruhiko; Shida, Yasuo; Tokuji, Yoshihiko

    2011-02-25

    Poly-trans-[(2-carboxyethyl) germasesquioxane], Ge-132 is a water-soluble organic germanium compound. Oral intake of dietary Ge-132 changes fecal color and we attempted to identify the fecal red pigment, which increased by the intake of dietary Ge-132. Sprague Dawley rats were given diets containing Ge-132 from 0 to 0.5% concentration. Fecal red pigment was extracted and purified for optical and structural studies. We examined the fecal red pigment content by high performance liquid chromatography (HPLC), and hepatic gene expressions relating to heme synthesis by reverse transcription polymerase chain reaction (RT-PCR). The purified red pigment had particular optical characteristics on the ultraviolet (UV)-visible spectrum (Soret band absorbance at 400 nm) and fluorescence emission at 600 nm by 400 nm excitation, and was identified as protoporphyrin IX by LC-MS analysis. Protoporphyrin IX significantly (P<0.05) increased 2.4-fold in the feces by the intake of a 0.5% Ge-132 diet. Gene expression analysis of the liver explained the increase of protoporphyrin IX by dietary Ge-132 as it enhanced (P<0.05) aminolevulinic acid synthase 1 (Alas1), a rate-limiting enzyme of heme synthesis, expression 1.8-fold, but decreased ferrochelatase (Fech) expression 0.6-fold (P<0.05). The results show that the intake of dietary Ge-132 is related to heme metabolism. Because protoporphyrin IX is used to treat chronic hepatitis, Ge-132 may be a beneficial substance to increase protoporphyrin IX in the liver.

  11. Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme.

    PubMed

    Bootorabi, Fatemeh; Jänis, Janne; Smith, Elona; Waheed, Abdul; Kukkurainen, Sampo; Hytönen, Vesa; Valjakka, Jarkko; Supuran, Claudiu T; Vullo, Daniela; Sly, William S; Parkkila, Seppo

    2010-08-01

    Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.

  12. A human transmembrane protein-tyrosine-phosphatase, PTP zeta, is expressed in brain and has an N-terminal receptor domain homologous to carbonic anhydrases.

    PubMed Central

    Krueger, N X; Saito, H

    1992-01-01

    Protein-tyrosine-phosphatases (PTPases, EC 3.1.3.48) play a crucial role in the regulation of protein tyrosine phosphorylation. Recently, it was found that the PTPase gene family exhibits a large variety of different functional domains associated with the PTPase catalytic domains. In this paper, we report the complete cDNA sequence of a human transmembrane PTPase, PTP zeta, isolated from fetal brain cDNA libraries. The deduced amino acid sequence of human PTP zeta is composed of a putative signal peptide of 19 amino acids, a very large extracellular domain of 1616 amino acids, a transmembrane peptide of 26 amino acids, and a cytoplasmic domain of 653 amino acids. The extracellular portion of human PTP zeta contains two striking structural features: the N-terminal 280-amino acid sequence that is homologous to carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1), and a sequence of 1048 amino acids without a cysteine residue. While it is unlikely that the carbonic anhydrase-like domain of PTP zeta has any carbonic anhydrase activity, its three-dimensional structure may be quite similar to that of carbonic anhydrases, a structure that appears ideal for binding a small soluble ligand. The cytoplasmic portion of human PTP zeta contains two repeated PTPase-like domains, which, when expressed in Escherichia coli, had PTPase activity in vitro. Mutational analyses indicate that only the membrane-proximal PTPase domain is catalytically active. Reverse transcription-polymerase chain reaction analyses indicate that human PTP zeta is highly expressed in a glioblastoma cell line. Images PMID:1323835

  13. Oral colonization by Streptococcus mutans and caries development is reduced upon deletion of carbonic anhydrase VI expression in saliva.

    PubMed

    Culp, David J; Robinson, Bently; Parkkila, Seppo; Pan, Pei-Wen; Cash, Melanie N; Truong, Helen N; Hussey, Thomas W; Gullett, Sarah L

    2011-12-01

    Carbonic anhydrase VI (CA VI), encoded by type A transcripts of the gene Car6, is a secretory product of salivary glands and is found in the enamel pellicle. Because higher caries prevalence is associated with lower salivary concentrations of CA VI in humans, we tested whether CA VI protects enamel surfaces from caries induced by Streptococcus mutans, using Car6(-/-) mice, in which salivary CA VI expression is absent. We detected aberrant Car6 type A transcripts in Car6(-/-) mice, likely targets for nonsense-mediated mRNA decay. Expression of the intracellular stress-induced isoform of CA VI encoded by type B transcripts was restricted to parotid and submandibular glands of wild type mice. The salivary function of Car6(-/-) mice was normal as assessed by the histology and protein/glycoprotein profiles of glands, salivary flow rates and protein/glycoprotein compositions of saliva. Surprisingly, total smooth surface caries and sulcal caries in Car6(-/-) mice were more than 6-fold and 2-fold lower than in wild type mice after infection with S. mutans strain UA159. Recoveries of S. mutans and total microbiota from molars were also lower in Car6(-/-) mice. To explore possible mechanisms for increased caries susceptibility, we found no differences in S. mutans adherence to salivary pellicles, in vitro. Interestingly, higher levels of Lactobacillus murinus and an unidentified Streptococcus species were cultivated from the oral microbiota of Car6(-/-) mice. Collective results suggest salivary CA VI may promote caries by modulating the oral microbiota to favor S. mutans colonization and/or by the enzymatic production of acid within plaque.

  14. Expression of active human blood clotting factor IX in transgenic mice: use of a cDNA with complete mRNA sequence.

    PubMed Central

    Choo, K H; Raphael, K; McAdam, W; Peterson, M G

    1987-01-01

    Haemophilia B is a bleeding disorder caused by a functional deficiency of the clotting factor IX. A full length human factor IX complementary DNA clone containing all the natural mRNA sequences plus some flanking intron sequences was constructed with a metallothionein promoter and introduced into transgenic mice by microinjection into the pronuclei of fertilised eggs. The transgenic mice expressed high levels of messenger RNA, gamma-carboxylated and glycosylated protein, and biological clotting activity that are indistinguishable from normal human plasma factor IX. This study demonstrates the feasibility of expressing highly complex heterologous proteins in transgenic mice. It also provides the groundwork for the production of large amounts of human factor IX in larger transgenic livestock for therapeutic use, and the investigation of alternative genetic therapies for haemophilia B. Images PMID:3029708

  15. Expression of carbonic anhydrase, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase in the lancelet Branchiostoma lanceolatum (Pallas, 1774).

    PubMed

    Pederzoli, Aurora; Mandrioli, Mauro; Mola, Lucrezia

    2014-04-01

    Sequencing of the amphioxus genome revealed that it contains a basic set of chordate genes involved in development and cell signaling. Despite the availability of genomic data, up till now no studies have been addressed on the comprehension of the amphioxus osmoregulation. Using primers designed on Branchiostoma floridae carbonic anhydrase (CA) II, cystic fibrosis transmembrane regulator (CFTR) and V-H(+)-ATPase, a 100bp long region, containing the protein region recognized by the respective antibodies, has been amplified and sequenced in B. lanceolatum indicating the presence of hortologous V-ATPase, CFTR and carbonic anhydrase II genes in Branchiostoma lanceolatum. Immunohistochemical results showed that all three transporting proteins are expressed in almost 90% of epithelial cells of the skin in B. lanceolatum adults with a different degree of positivity in different regions of body wall and with a different localization in the cells. The comparison of results between young and adult lancelets showed that the distribution of these transporters is quite different. Indeed, in the young specimens the expression pattern of all tested molecules appears concentrated at the gut level, whereas in adult the gut loses its key role that is mostly supported by skin.

  16. Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-[1,2,3]triazolo[4,5d]pyrimidin 6(7H)yl)urea.

    PubMed

    Kumari, Shikha; Idrees, Danish; Mishra, Chandra Bhushan; Prakash, Amresh; Wahiduzzaman; Ahmad, Faizan; Hassan, Md Imtaiyaz; Tiwari, Manisha

    2016-03-01

    Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug design and discovery process. Here, we performed fluorescence-binding studies and inhibition assay combined with molecular docking and molecular dynamics (MD) simulation analyses to determine the binding affinity of two synthesized triazolo-pyrimidine urea derived (TPUI and TPUII) compounds with CAIX and CAII. Fluorescence binding results are showing that molecule TPUI has an excellent binding-affinity for CAIX (kD=0.048μM). The TPUII also exhibits an appreciable binding affinity (kD=7.52μM) for CAIX. TPUI selectively inhibits CAIX as compared to TPUII in the 4-NPA assay. Docking studies show that TPUI is spatially well-fitted in the active site cavity of CAIX, and is involve in H-bond interactions with His94, His96, His119, Thr199 and Thr200. MD simulation studies revealed that TPUI efficiently binds to CAIX and essential active site residual interaction is consistent during the entire simulation of 40ns. These studies suggest that TPUI appeared as novel class of CAIX inhibitor, and may be used as a lead molecule for the development of potent and selective CAIX inhibitor for the hypoxia-induced cancer therapy.

  17. Transient accumulation of Mg-protoporphyrin IX regulates expression of PhANGs - New evidence for the signaling role of tetrapyrroles in mature Arabidopsis plants.

    PubMed

    Zhang, Zhong-Wei; Yuan, Shu; Feng, Hong; Xu, Fei; Cheng, Jian; Shang, Jing; Zhang, Da-Wei; Lin, Hong-Hui

    2011-05-01

    Genetic and physiological studies have revealed evidence for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis associated nuclear genes (PhANGs). It has been proposed that the tetrapyrrole pathway intermediate Mg-protoporphyrin IX (Mg-proto IX) acts as the signaling molecule in the pathways and accumulates in the chloroplasts and cytosol of the cell after treatment with the herbicide Norflurazon (NF). However, the role of Mg-Proto IX in plastid signaling has been challenged by two recent reports. In this paper, new evidence is presented supporting Mg-Proto IX as a plastid-signaling molecule in mature Arabidopsis seedlings. Fluorescence HPLC and confocal microscope observation verified that a short-term (<96h) NF treatment resulted in a large accumulation of Mg-Proto IX accompanying with Lhcb repression, whereas the long-term NF treatments caused marked changes of tetrapyrrole pools, while Lhcb expression was continuously repressed. These results may explain the discrepancies among different reports. Reactive oxygen species (ROS) eliminator treatments only partly reversed the NF-induced repression of Lhcb. Therefore, the NF generates both ROS signals and Mg-Proto IX signals. Furthermore, our data suggested that plastid signal transduction through plastid GUN1 protein is independent of tetrapyrrole export from the plastid.

  18. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  19. An immunohistochemical study of the expression of the hypoxia markers Glut-1 and Ca-IX in canine sarcomas.

    PubMed

    Abbondati, E; Del-Pozo, J; Hoather, T M; Constantino-Casas, F; Dobson, J M

    2013-11-01

    Tumor hypoxia has been associated with increased malignancy, likelihood of metastasis, and increased resistance to radiotherapy and chemotherapy in human medicine. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that is induced by tumor hypoxia and regulates the pathways involved in cellular response and adaptation to the hostile tumor microenvironment. HIF-1 induces transcription of different proteins, including Ca-IX and Glut-1, which are considered endogenous markers of chronic hypoxia in solid tumors in humans. In this study, sections from 40 canine sarcomas (20 histiocytic sarcomas and 20 low-grade soft-tissue sarcomas) were immunostained for these markers. Expression of Glut-1 was scored based on percentage of positive staining cells (0 = <1%; 1 = 1%-50%; 2 = >50%) and intensity of cellular staining (1 = weak; 2 = strong); Ca-IX was scored based on percentage of positive cells (0 = <1%; 1 = 1%-30%; 2 = >30%). Intratumoral microvessel density was measured using CD31 to assess intratumoral neoangiogenesis. Histiocytic sarcomas showed statistically significant higher Glut-1 immunoreactivity and angiogenesis than did low-grade soft-tissue sarcomas. Intratumoral microvessel density in histiocytic sarcomas was positively associated with Glut-1 immunoreactivity score. These findings suggest a potential role of hypoxia in the biology of these tumors and may provide a base for investigation of the potential prognostic use of these markers in naturally occurring canine tumors.

  20. Hemin and Magnesium-Protoporphyrin IX Induce Global Changes in Gene Expression in Chlamydomonas reinhardtii[C][W

    PubMed Central

    Voß, Björn; Meinecke, Linda; Kurz, Thorsten; Al-Babili, Salim; Beck, Christoph F.; Hess, Wolfgang R.

    2011-01-01

    Retrograde signaling is a pathway of communication from mitochondria and plastids to the nucleus in the context of cell differentiation, development, and stress response. In Chlamydomonas reinhardtii, the tetrapyrroles magnesium-protoporphyrin IX and heme are only synthesized within the chloroplast, and they have been implicated in the retrograde control of nuclear gene expression in this unicellular green alga. Feeding the two tetrapyrroles to Chlamydomonas cultures was previously shown to transiently induce five nuclear genes, three of which encode the heat shock proteins HSP70A, HSP70B, and HSP70E. In contrast, controversial results exist on the possible role of magnesium-protoporphyrin IX in the repression of genes for light-harvesting proteins in higher plants, raising the question of how important this mode of regulation is. Here, we used genome-wide transcriptional profiling to measure the global impact of these tetrapyrroles on gene regulation and the scope of the response. We identified almost 1,000 genes whose expression level changed transiently but significantly. Among them were only a few genes for photosynthetic proteins but several encoding enzymes of the tricarboxylic acid cycle, heme-binding proteins, stress-response proteins, as well as proteins involved in protein folding and degradation. More than 50% of the latter class of genes was also regulated by heat shock. The observed drastic fold changes at the RNA level did not correlate with similar changes in protein concentrations under the tested experimental conditions. Phylogenetic profiling revealed that genes of putative endosymbiontic origin are not overrepresented among the responding genes. This and the transient nature of changes in gene expression suggest a signaling role of both tetrapyrroles as secondary messengers for adaptive responses affecting the entire cell and not only organellar proteins. PMID:21148414

  1. Molecular cloning and sequence analysis of two carbonic anhydrase in the swimming crab Portunus trituberculatus and its expression in response to salinity and pH stress.

    PubMed

    Pan, Luqing; Hu, Dongxu; Liu, Maoqi; Hu, Yanyan; Liu, Shengnan

    2016-01-15

    Carbonic anhydrase (CA) is involved in ion transport, acid-base balance and pH regulation by catalyzing the interconversion of CO2 and HCO3(-). In this study, full-length cDNA sequences of two CA isoforms were identified from Portunus trituberculatus. One was Portunus trituberculatus cytoplasmic carbonic anydrase (PtCAc) and the other one was Portunus trituberculatus glycosyl-phosphatidylinositol-linked carbonic anhydrase (PtCAg). The sequence of PtCAc was formed by an ORF of 816 bp, encoding a protein of 30.18 kDa. The PtCAg was constituted by an ORF of 927 bp, encoding a protein of 34.09 kDa. The deduced amino acid sequences of the two CA isoforms were compared to other crustacean' CA sequences. Both of them reflected high conservation of the residues and domains essential to the function of the two enzymes. The tissue expression analysis of PtCAc and PtCAg were detected in gill, muscle, hepatopancreas, hemocytes and gonad. PtCAc and PtCAg gene expressions were studied under salinity and pH challenge. The results showed that when salinity decreased (30 to 20 ppt), the mRNA expression of PtCAc increased significantly at 24 and 48 h, and the highest value appeared at 24h. The mRNA expression of PtCAg had the same situation with PtCAc. However, when salinity increased (30 to 35 ppt), only the mRNA expression of PtCAc increased significantly at 48 h. When pH changed, only the mRNA expression of PtCAc increased significantly at 12h, which was under low pH situation. The mRNA expression of PtCAg increased significantly at 12-48 h, and there was no significant difference of the expression between the pH challenged group and the control group in other experimental time. The results provided the base of understanding CA' function and the underlying mechanism in response to environmental changes in crustaceans.

  2. Plant Carbonic Anhydrases

    PubMed Central

    Atkins, C. A.; Patterson, B. D.; Graham, D.

    1972-01-01

    On the basis of polyacrylamide gradient gel electrophoresis of leaf extracts from 24 species of higher plants, two main forms of carbonic anhydrase (EC 4.2.1.1) were recognized; the “dicotyledon” type and the “monocotyledon” type. More than one band of enzyme was found on gels from most species, suggesting the possibility of carbonic anhydrase isoenzymes in higher plants. Images PMID:16658144

  3. Increased expression of mitochondrial benzodiazepine receptors following low-level light treatment facilitates enhanced protoporphyrin IX production in glioma-derived cells in vitro

    NASA Astrophysics Data System (ADS)

    Bisland, S. K.; Hassanali, N. S.; Johnson, C.; Wilson, B. C.

    2007-02-01

    This study investigates whether low level light treatment (LLLT) can enhance the expression of Peripheral-type mitochondrial benzodiazepine receptors (PBRs) on the glioma-derived tumour cell line, CNS-1, and by doing so promote the synthesis of protoporphyrin IX (PpIX) and increase the photodynamic therapy (PDT)-induced cell kill using 5-aminolevulinic acid (ALA). The endogenous photosensitizer, (PpIX) and related metabolites including coproporphyrin III are known to traffic via the PBRs on the outer mitochondrial membrane on their passage into or out of the mitochondria. Astrocyte-derived cells within the brain express PBRs, while neurons express the central-type of benzodiazepine receptor. CNS-1 cells were exposed to a range of differing low-level light protocols immediately prior to PDT. LLLT involved using broad-spectrum light or monochromatic laser light specific to 635 or 905 nm wavelength. Cells (5μ10 5) were exposed to a range of LLLT doses (0, 1 or 5 J/cm2) using a fixed intensity of 10 mW/cm2 and subsequently harvested for cell viability, immunofluorescence or western blot analysis of PBR expression. The amount of PpIX within the cells was determined using chemical extraction techniques. Results confirm the induction of PBR following LLLT is dependent on the dose and wavelength of light used. Broadspectrum light provided the greatest cell kill following PDT, although LLLT with 635 nm or 905 nm also increased cell kill as compared to PDT alone. All LLLT regimens increased PBR expression compared to controls with corresponding increases in PpIX production. These data suggest that by selectively increasing PBR expression in tumour cells, LLLT may facilitate enhanced cell kill using ALA-PDT without damaging surrounding normal brain.

  4. P-glycoprotein-mediated chemoresistance is reversed by carbonic anhydrase XII inhibitors

    PubMed Central

    Kopecka, Joanna; Rankin, Gregory M.; Salaroglio, Iris C.

    2016-01-01

    Carbonic anhydrase XII (CAXII) is a membrane enzyme that maintains pH homeostasis and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Here, we investigated a panel of eight CAXII inhibitors (compounds 1–8), for their potential to reverse Pgp mediated tumor cell chemoresistance. Inhibitors (5 nM) were screened in human and murine cancer cells (colon, lung, breast, bone) with different expression levels of CAXII and Pgp. We identified three CAXII inhibitors (compounds 1, 2 and 4) that significantly (≥ 2 fold) increased the intracellular retention of the Pgp-substrate and chemotherapeutic doxorubicin, and restored its cytotoxic activity. The inhibitors lowered intracellular pH to indirectly impair Pgp activity. Ca12-knockout assays confirmed that the chemosensitizing property of the compounds was dependent on active CAXII. Furthermore, in a preclinical model of drug-resistant breast tumors compound 1 (1900 ng/kg) restored the efficacy of doxorubicin to the same extent as the direct Pgp inhibitor tariquidar. The expression of carbonic anhydrase IX had no effect on the intracellular doxorubicin accumulation. Our work provides strong evidence that CAXII inhibitors are effective chemosensitizer agents in CAXII-positive and Pgp-positive cancer cells. The use of CAXII inhibitors may represent a turning point in combinatorial chemotherapeutic schemes to treat multidrug-resistant tumors. PMID:27811376

  5. Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance

    PubMed Central

    Feng, Han-Zhong; Jin, J.-P.

    2016-01-01

    Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies. PMID:28018233

  6. Carbonic Anhydrase III Is Expressed in Mouse Skeletal Muscles Independent of Fiber Type-Specific Myofilament Protein Isoforms and Plays a Role in Fatigue Resistance.

    PubMed

    Feng, Han-Zhong; Jin, J-P

    2016-01-01

    Carbonic anhydrase III (CAIII) is a metabolic enzyme and a regulator for intracellular pH. CAIII has been reported with high level expression in slow twitch skeletal muscles. Here we demonstrate that CAIII is expressed in multiple slow and fast twitch muscles of adult mouse independent of the expression of myosin isoforms. Expressing similar fast type of myofilament proteins, CAIII-positive tibial anterior (TA) muscle exhibits higher tolerance to fatigue than that of CAIII-negative fast twitch extensor digitorum longus (EDL) muscle in in situ contractility studies. We further studied the muscles of CAIII knockout (Car3-KO) mice. The loss of CAIII in soleus and TA muscles in Car3-KO mice did not change muscle mass, sarcomere protein isoform contents, and the baseline twitch and tetanic contractility as compared with age-matched wild type (WT) controls. On the other hand, Car3-KO TA muscle showed faster force reduction at the beginning but higher resistance at the end during a fatigue test, followed by slower post fatigue recovery than that of WT TA muscle. Superfused Car3-KO soleus muscle also had faster total force reduction during fatigue test than that of WT soleus. However, it showed a less elevation of resting tension followed by a better post fatigue recovery under acidotic stress. CAIII was detected in neonatal TA and EDL muscle, downregulated during development, and then re-expressed in adult TA but not EDL muscles. The expression of CAIII in Tnnt1-KO myopathy mouse soleus muscle that has diminished slow fiber contents due to the loss of slow troponin T remained high. Car3-KO EDL, TA, and soleus muscles showed no change in the expression of mitochondria biomarker proteins. The data suggest a fiber type independent expression of CAIII with a role in the regulation of intracellular pH in skeletal muscle and may be explored as a target for improving fatigue resistance and for the treatment of TNNT1 myopathies.

  7. 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.

    PubMed

    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.

  8. CO2 reduction and organic compounds production by photosynthetic bacteria with surface displayed carbonic anhydrase and inducible expression of phosphoenolpyruvate carboxylase.

    PubMed

    Park, Ju-Yong; Kim, Yang-Hoon; Min, Jiho

    2017-01-01

    In Rhodobacter sphaeroides, carbonic anhydrase (CA; EC 4.2.1.1) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-) while phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), an enzyme involved in the carbon metabolism that catalyzed the fixation of CO2 to PEP, is a key factor for biological fixation of CO2 and enhances the production of organic compounds. In this study, the recombinant R. sphaeroides with highly-expressed CA was developed based on a surface displayed system of CA (pJY-OmpCA) on the outer membrane of R. sphaeroides using outer membrane protein (Omp) in R. sphaeroides, Finally, two more different recombinant R. sphaeroides were developed, which transformed with a two-vector system harboring cytosolic expressed CA (pJY-OmpCA-CA)or PEPC (pJY-OMPCA-PEPC) in R. sphaeroides with surface displayed CA on the outer membrane. In case of recombinant R. sphaeroides with the pJY-OmpCA-PEPC, it has shown the highest CO2 reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid). It means that the surface displayed CA on the R. sphaeroides would accelerate the CO2-bicabonate conversion on the bacterial outer membrane. Moreover, inducible over-expression of PEPC with surface-displayed CA was successfully used to facilitate a rapider CO2 reduction and quicker production of organic compounds.

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

    PubMed

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

    2016-12-01

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

  10. Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

    PubMed

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

    2016-06-03

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

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

    PubMed

    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.

  12. Pairwise Comparison of 89Zr- and 124I-labeled cG250 Based on Positron Emission Tomography Imaging and Non-Linear Immunokinetic Modeling: In Vivo Carbonic Anhydrase IX Receptor Binding and Internalization in Mouse Xenografts of Clear Cell Renal Carcinoma

    PubMed Central

    Cheal, Sarah M.; Punzalan, Blesida; Doran, Michael G.; Evans, Michael J.; Osborne, Joseph R.; Lewis, Jason S.; Zanzonico, Pat; Larson, Steven M.

    2014-01-01

    Purpose The positron-emitting tomography (PET) tracer, 124I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for pre-surgical diagnosis of clear renal cell carcinoma (cRCC) [1, 2]. The radiometal zirconium-89 (89Zr), however, may offer advantages as a surrogate PET nuclide over 124I in terms of greater tumor uptake and retention [3]. In the current report, we have developed a non-linear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between 124I-cG250 and 89Zr- cG250 using a human cRCC xenograft tumor model in mice. We believe that his unique model better relates quantitative imaging data to the salient biologic features of tumor antibody-antigen binding and turnover. Methods We conducted experiments with 89Zr-cG250 and 124I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial-PET imaging was performed in mice bearing sub-cutaneous cRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumor were derived from the PET images using non-linear compartmental modeling. Results The two tracers demonstrate virtually identical tumor-cell binding and internalization but with markedly different retentions in vitro. Superior PET images were obtained using 89Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous wash-out from normal tissues. Estimates of cG250-CAIX complex turnover were 1.35–5.51 × 1012 molecules per hour per gram of tumor (20% of receptors internalized per hour), and the ratio of 124I/89Zr atoms released per unit time by tumor was 17.5. Conclusions Pairwise evaluation of 89Zr-cG250 and 124I-cG250 provided the basis for a non-linear immunokinetic model which yielded quantitative information about

  13. Effect of CO{sub 2} concentration on carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase expression in pea

    SciTech Connect

    Majeau, N.; Coleman, J.R.

    1996-10-01

    The effect of external CO{sub 2} concentration on the expression of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in pea (Pisum sativum cv Little Marvel) leaves. Enzyme activities and their transcript levels were reduced in plants grown at 1000 {mu}L/L CO{sub 2} compared with plants grown in ambient air. Growth at 160 {mu}L/L CO{sub 2} also appeared to reduce steady-state transcript levels for the rbcS, the gene encoding the small subunit of Rubisco, and for ca, the gene encoding CA; however, rbcS transcripts were reduced to a greater extent at this concentration. Rubisco activity was slightly lower in plants grown at 160 {mu}L/L CO{sub 2}, and CA activity was significantly higher than that observed in air-grown plants. Transfer of plants from 1000 {mu}L/L to air levels of CO{sub 2} resulted in a rapid increase in both ca and rbcS transcript abundance in fully expanded leaves, followed by an increase in enzyme activity. Plants transferred from air to high-CO{sub 2} concentrations appeared to modulate transcript abundance and enzyme activity less quickly. Foliar carbohydrate levels were also examined in plants grown continuously at high and ambient CO{sub 2}, and following changes in growth conditions that rapidly altered ca and rbcS transcript abundance and enzyme activities. 39 refs., 2 figs., 3 tabs.

  14. Expression and characterization of a codon-optimized alkaline-stable carbonic anhydrase from Aliivibrio salmonicida for CO2 sequestration applications.

    PubMed

    Jun, So-Young; Kim, Sung Ho; Kanth, Bashista Kumar; Lee, Jinwon; Pack, Seung Pil

    2017-03-01

    The CO2 mineralization process, accelerated by carbonic anhydrase (CA) was proposed for the efficient capture and storage of CO2, the accumulation of which in the atmosphere is the main cause of global warming. Here, we characterize a highly stable form of the cloned CA from the Gram-negative marine bacterium Aliivibrio salmonicida, named ASCA that can promote CO2 absorption in an alkaline solvent required for efficient carbon capture. We designed a mature form of ASCA (mASCA) using a codon optimization of ASCA gene and removal of ASCA signal peptide. mASCA was highly expressed (255 mg/L) with a molecular weight of approximately 26 kDa. The mASCA enzyme exhibited stable esterase activity within a temperature range of 10-60 °C and a pH range of 6-11. mASCA activity remained stable for 48 h at pH 10. We also investigated its inhibition profiles using inorganic anions, such as acetazolamide, sulfanilamide, iodide, nitrate, and azide. We also demonstrate that mASCA is capable of catalyzing the conversion of CO2 to CaCO3 (calcite form) in the presence of Ca(2+). It should be noted that mASCA enzyme exhibits high production yield and sufficient stabilities against relatively high temperature and alkaline pH, which are required conditions for the development of more efficient enzymatic CCS systems.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-21

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

  17. Expression and purification of TAT-fused carbonic anhydrase III and its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation

    PubMed Central

    Shang, Xiliang; Bao, Yuanyuan; Ren, Huimin; Huang, He; Li, Yunxia

    2012-01-01

    Introduction Carbonic anhydrase III (CAIII) is remarkably abundant in slow skeletal muscles. It has multiple biological activities which could dissipate or resist some fatigue-related substances. In this study, we purified trans-activating transcriptional activator (TAT) fused CAIII protein and investigated its effect on C2C12 cell apoptosis induced by hypoxia/reoxygenation. Material and methods The CAIII and TAT-CAIII genes were constructed, cloned into plasmid pET28a and expressed in Escherichia coli BL21 (DE3). The fusion proteins were purified with a nickel-nitrilotriacetic acid affinity chromatography column and then verified by Western blot and phosphatase activity staining subsequently. The C2C12 cells were treated respectively with serum-free medium containing 1 μM TAT-CAIII or 1 μM CAIII for 1 h and the intracellular distributions of fusion proteins were observed by indirect immunofluorescence. The effect of TAT-CAIII on C2C12 cell apoptosis induced by hypoxia/reoxygenation was detected by flow cytometry. Results The CAIII and TAT-CAIII fusion proteins were expressed and purified successfully. After being cultured for 1 h, green fluorescence was visible in TAT-CAIII group cells under the fluorescence microscope, while no fluorescence was found in the CAIII group. Compared with the oxygen-glucose deprivation group, the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation in the TAT-CAIII group decreased significantly (p < 0.001). Conclusions The purified TAT-CAIII could be transferred into cells efficiently and clearly decreased the apoptosis rate of C2C12 cells induced by hypoxia/reoxygenation, which indicated that it had antioxidative activity. This study lays an experimental basis for future research on the relationship between CAIII and muscle fatigue. PMID:23056085

  18. A two-component system regulates gene expression of the type IX secretion component proteins via an ECF sigma factor

    PubMed Central

    Kadowaki, Tomoko; Yukitake, Hideharu; Naito, Mariko; Sato, Keiko; Kikuchi, Yuichiro; Kondo, Yoshio; Shoji, Mikio; Nakayama, Koji

    2016-01-01

    The periodontopathogen Porphyromonas gingivalis secretes potent pathogenic proteases, gingipains, via the type IX secretion system (T9SS). This system comprises at least 11 components; however, the regulatory mechanism of their expression has not yet been elucidated. Here, we found that the PorY (PGN_2001)-PorX (PGN_1019)-SigP (PGN_0274) cascade is involved in the regulation of T9SS. Surface plasmon resonance (SPR) analysis revealed a direct interaction between a recombinant PorY (rPorY) and a recombinant PorX (rPorX). rPorY autophosphorylated and transferred a phosphoryl group to rPorX in the presence of Mn2+. These results demonstrate that PorX and PorY act as a response regulator and a histidine kinase, respectively, of a two component system (TCS), although they are separately encoded on the chromosome. T9SS component-encoding genes were down-regulated in a mutant deficient in a putative extracytoplasmic function (ECF) sigma factor, PGN_0274 (SigP), similar to the porX mutant. Electrophoretic gel shift assays showed that rSigP bound to the putative promoter regions of T9SS component-encoding genes. The SigP protein was lacking in the porX mutant. Co-immunoprecipitation and SPR analysis revealed the direct interaction between SigP and PorX. Together, these results indicate that the PorXY TCS regulates T9SS-mediated protein secretion via the SigP ECF sigma factor. PMID:26996145

  19. Embryonic exposure to o,p'-DDT causes eggshell thinning and altered shell gland carbonic anhydrase expression in the domestic hen.

    PubMed

    Holm, Lena; Blomqvist, Alexandra; Brandt, Ingvar; Brunström, Björn; Ridderstråle, Yvonne; Berg, Cecilia

    2006-10-01

    The mechanism for contaminant-induced eggshell thinning in wild birds remains to be clarified. It is generally assumed, however, that it results from exposure of the adult laying female. We have reported that embryonic exposure to the synthetic estrogen ethynylestradiol (EE2) results in eggshell thinning in the domestic hen. The objective of this study was to investigate whether eggshell thinning can be induced following in ovo exposure to a bioaccumulating estrogenic environmental contaminant, o,p'-DDT. Ethynylestradiol was used as a positive control. Domestic hens exposed in ovo to o,p'-DDT (37 or 75 microg/g egg) or EE2 (60 ng/g egg) laid eggs with thinner shells than the control birds. The hens from these exposure groups also had a significantly reduced frequency of shell gland capillaries with carbonic anhydrase (CA) activity, a key enzyme in eggshell formation. The decreased number of capillaries with CA activity suggests that a developmentally induced disruption of CA expression in the shell gland was involved in the eggshell thinning found in this study. Egg laying was not affected in hens exposed embryonically to 37 or 75 microg o,p'-DDT/g egg, whereas it was inhibited in hens exposed to higher doses. Decreased lengths of the left oviduct and its infundibulum were seen after embryonic treatment with o,p'-DDT or EE2. In addition, o,p'-DDT exposure resulted in right oviduct retention. The results support our hypothesis that eggshell thinning in avian wildlife can result from a functional malformation in the shell gland, induced by embryonic exposure to estrogenic substances.

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

    PubMed

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

    2014-07-01

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

  1. In vitro evaluation of ESE-15-ol, an estradiol analogue with nanomolar antimitotic and carbonic anhydrase inhibitory activity.

    PubMed

    Stander, Barend Andre; Joubert, Fourie; Tu, Chingkuang; Sippel, Katherine H; McKenna, Robert; Joubert, Annie Margaretha

    2012-01-01

    Antimitotic compounds are still one of the most widely used chemotherapeutic anticancer drugs in the clinic today. Given their effectiveness against cancer it is beneficial to continue enhancing these drugs. One way is to improve the bioavailability and efficacy by synthesizing derivatives that reversibly bind to carbonic anhydrase II (CAII) in red blood cells followed by a slow release into the blood circulation system. In the present study we describe the in vitro biological activity of a reduced derivative of 2-ethyl-3-O-sulphamoyl-estradiol (2EE), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol). ESE-15-ol is capable of inhibiting carbonic anhydrase activity in the nanomolar range and is selective towards a mimic of carbonic anhydrase IX when compared to the CAII isoform. Docking studies using Autodock Vina suggest that the dehydration of the D-ring plays a role towards the selectivity of ESE-15-ol to CAIX and that the binding mode of ESE-15-ol is substantially different when compared to 2EE. ESE-15-ol is able to reduce cell growth to 50% after 48 h at 50-75 nM in MCF-7, MDA-MB-231, and MCF-12A cells. The compound is the least potent against the non-tumorigenic MCF-12A cells. In vitro mechanistic studies demonstrate that the newly synthesized compound induces mitochondrial membrane depolarization, abrogates the phosphorylation status of Bcl-2 and affects gene expression of genes associated with cell death and mitosis.

  2. Title IX Resource Guide

    ERIC Educational Resources Information Center

    Office for Civil Rights, US Department of Education, 2015

    2015-01-01

    Title IX of the Education Amendments of 1972 (Title IX) prohibits discrimination based on sex in education programs and activities in federally funded schools at all levels. If any part of a school district or college receives any Federal funds for any purpose, all of the operations of the district or college are covered by Title IX. The essence…

  3. Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors

    PubMed Central

    Koeberl, Dwight D.; Alexander, Ian E.; Halbert, Christine L.; Russell, David W.; Miller, A. Dusty

    1997-01-01

    We previously found that gene transduction by adeno-associated virus (AAV) vectors in cell culture can be stimulated over 100-fold by treatment of the target cells with agents that affect DNA metabolism, such as irradiation or topoisomerase inhibitors. Here we show that previous γ-irradiation increased the transduction rate in mouse liver by up to 900-fold, and the topoisomerase inhibitor etoposide increased transduction by about 20-fold. Similar rates of hepatic transduction were obtained by direct injection of the liver or by systemic delivery via tail vein injection. Hepatocytes were much more efficiently transduced than other cells after systemic delivery, and up to 3% of all hepatocytes could be transduced after one vector injection. The presence of wild-type AAV, which contaminates many AAV vector preparations, was required to observe a full response to γ-irradiation. Injection of mice with AAV vectors encoding human clotting factor IX after γ-irradiation resulted in synthesis of low levels of human clotting factor IX for the 5-month period of observation. These studies show the potential of targeted gene transduction of the liver by AAV vectors for treatment of various hematological or metabolic diseases. PMID:9037069

  4. Molecular basis of human carbonic anhydrase II deficiency.

    PubMed Central

    Roth, D E; Venta, P J; Tashian, R E; Sly, W S

    1992-01-01

    Deficiency of carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) is the primary defect in the syndrome of osteopetrosis, renal tubular acidosis, and cerebral calcification. In this report we describe the molecular basis for carbonic anhydrase II deficiency in the American family in which the association of carbonic anhydrase II deficiency with this syndrome was first recognized. The three affected siblings from this family are compound heterozygotes, each having inherited two different mutations in the structural gene for carbonic anhydrase II. The paternal mutation is a splice acceptor site mutation at the 3' end of intron 5. The maternal mutation is a missense mutation in exon 3 that substitutes a tyrosine for histidine-107. We show that the mutant enzyme expressed in bacteria from the cDNA containing the His-107----Tyr mutation has detectable, though greatly reduced, activity. We suggest that residual activity of the His-107----Tyr mutant enzyme may explain the absence of mental retardation and the relatively mild phenotype of carbonic anhydrase II deficiency in affected members of this family. Images PMID:1542674

  5. Genetics Home Reference: carbonic anhydrase VA deficiency

    MedlinePlus

    ... people with carbonic anhydrase VA deficiency have excess ammonia in the blood (hyperammonemia), problems with acid-base ... anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism. Proc Natl Acad Sci ...

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

    PubMed Central

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

    2016-01-01

    Carbonic anhydrase plays a key role in CO2 transport, acid-base and ion regulation and metabolic processes in vertebrates. While several carbonic anhydrase isoforms have been identified in numerous vertebrate species, basal lineages such as the cyclostomes have remained largely unexamined. Here we investigate the repertoire of cytoplasmic carbonic anhydrases in the sea lamprey (Petromyzon marinus), that has a complex life history marked by a dramatic metamorphosis from a benthic filter-feeding ammocoete larvae into a parasitic juvenile which migrates from freshwater to seawater. We have identified a novel carbonic anhydrase gene (ca19) beyond the single carbonic anhydrase gene (ca18) that was known previously. Phylogenetic analysis and synteny studies suggest that both carbonic anhydrase genes form one or two independent gene lineages and are most likely duplicates retained uniquely in cyclostomes. Quantitative PCR of ca19 and ca18 and protein expression in gill across metamorphosis show that the ca19 levels are highest in ammocoetes and decrease during metamorphosis while ca18 shows the opposite pattern with the highest levels in post-metamorphic juveniles. We propose that a unique molecular switch occurs during lamprey metamorphosis resulting in distinct gill carbonic anhydrases reflecting the contrasting life modes and habitats of these life-history stages. PMID:27703170

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

    USGS Publications Warehouse

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

  9. A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects.

    PubMed

    Carta, Fabrizio; Di Cesare Mannelli, Lorenzo; Pinard, Melissa; Ghelardini, Carla; Scozzafava, Andrea; McKenna, Robert; Supuran, Claudiu T

    2015-04-15

    A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known, and some appear to be involved in pain. These sulfonamides showed modest inhibition against the cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain relieving effect, a fact never observed earlier. This is the first report of rationally designed sulfonamide CA inhibitors with pain effective modulating effects.

  10. Polyamines and α-Carbonic Anhydrases.

    PubMed

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

    2016-12-15

    Natural products represent a straightforward source for molecular structures bearing a vast array of chemical features and potentially useful for biomedical purposes. Recent examples of this type include the discovery of the coumarins and the polyamine natural products as atypical chemotypes for the inhibition of the metalloenzymes carbonic anhydrases (CAs; EC 4.2.2.1). CA enzymes are established pharmacological targets for important pathologies, which, among others, include glaucoma, hypoxic tumors, and central nervous system (CNS)-affecting diseases. Moreover, they are expressed in many bacteria, fungi and helminths which are the etiological agents of the majority of infectious diseases. In this context, natural products represent the ideal source of new and selective druggable CA modulators for biomedical purposes. Herein we report the state of the art on polyamines of natural origin as well as of synthetic derivatives as inhibitors of human CAs.

  11. Association of Reduced Type IX Collagen Gene Expression in Human Osteoarthritic Chondrocytes With Epigenetic Silencing by DNA Hypermethylation

    PubMed Central

    Imagawa, Kei; de Andrés, María C; Hashimoto, Ko; Itoi, Eiji; Otero, Miguel; Roach, Helmtrud I; Goldring, Mary B; Oreffo, Richard O C

    2014-01-01

    Objective To investigate whether the changes in collagen gene expression in osteoarthritic (OA) human chondrocytes are associated with changes in the DNA methylation status in the COL2A1 enhancer and COL9A1 promoter. Methods Expression levels were determined using quantitative reverse transcription–polymerase chain reaction, and the percentage of DNA methylation was quantified by pyrosequencing. The effect of CpG methylation on COL9A1 promoter activity was determined using a CpG-free vector; cotransfections with expression vectors encoding SOX9, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α were carried out to analyze COL9A1 promoter activities in response to changes in the methylation status. Chromatin immunoprecipitation assays were carried out to validate SOX9 binding to the COL9A1 promoter and the influence of DNA methylation. Results Although COL2A1 messenger RNA (mRNA) levels in OA chondrocytes were 19-fold higher than those in the controls, all of the CpG sites in the COL2A1 enhancer were totally demethylated in both samples. The levels of COL9A1 mRNA in OA chondrocytes were 6,000-fold lower than those in controls; 6 CpG sites of the COL9A1 promoter were significantly hypermethylated in OA patients as compared with controls. Treatment with 5-azadeoxycitidine enhanced COL9A1 gene expression and prevented culture-induced hypermethylation. In vitro methylation decreased COL9A1 promoter activity. Mutations in the 5 CpG sites proximal to the transcription start site decreased COL9A1 promoter activity. Cotransfection with SOX9 enhanced COL9A1 promoter activity; CpG methylation attenuated SOX9 binding to the COL9A1 promoter. Conclusion This first demonstration that hypermethylation is associated with down-regulation of COL9A1 expression in OA cartilage highlights the pivotal role of epigenetics in OA, involving not only hypomethylation, but also hypermethylation, with important therapeutic implications for OA treatment. PMID:25048791

  12. Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

    PubMed

    Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

    2016-05-01

    Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.

  13. Intercollegiate Athletics and Title IX

    ERIC Educational Resources Information Center

    Cox, Thomas A.

    1977-01-01

    The application of Title IX and the HEW regulation to intercollegiate sports is described, and the relationships among Title IX, the equal protection doctrine, and the proposed equal rights amendment to the Constitution are assessed. (LBH)

  14. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    PubMed Central

    Balestra, Dario; Scalet, Daniela; Pagani, Franco; Rogalska, Malgorzata Ewa; Mari, Rosella; Bernardi, Francesco; Pinotti, Mirko

    2016-01-01

    In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G) or 3′ (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients. PMID:27701399

  15. Dendrimers incorporating benzenesulfonamide moieties strongly inhibit carbonic anhydrase isoforms I-XIV.

    PubMed

    Carta, Fabrizio; Osman, Sameh M; Vullo, Daniela; AlOthman, Zeid; Supuran, Claudiu T

    2015-06-21

    As extension of our previous study herein we report a comprehensive investigation of poly(amidoamine) (PAMAM) dendrimers as modulators of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I-XIV. Interestingly inhibitory activity was observed for the non-functionalized dendrimers against the hCA I, VII, IX, XII and XIV isoforms, whereas activation properties were reported only for the cytosolic abundant hCA II. Highly efficient inhibitory action against many isoforms having medicinal chemistry applications, such as hCA II, V, VII, IX, XII and XIV, was observed for the PAMAM functionalized counterparts bearing 4, 8, 16 and 32 benzenesulfonamide moieties. Possible applications of dendrimer-CA inhibitors as therapeutic/diagnostic agents are envisaged.

  16. Exonuclease IX of Escherichia coli.

    PubMed Central

    Shafritz, K M; Sandigursky, M; Franklin, W A

    1998-01-01

    The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways. PMID:9592142

  17. Major role of local immune responses in antibody formation to factor IX in AAV gene transfer.

    PubMed

    Wang, L; Cao, O; Swalm, B; Dobrzynski, E; Mingozzi, F; Herzog, R W

    2005-10-01

    The risk of an immune response to the coagulation factor IX (F.IX) transgene product is a concern in gene therapy for the X-linked bleeding disorder hemophilia B. In order to investigate the mechanism of F.IX-specific lymphocyte activation in the context of adeno-associated viral (AAV) gene transfer to skeletal muscle, we injected AAV-2 vector expressing human F.IX (hF.IX) into outbred immune-competent mice. Systemic hF.IX levels were transiently detected in the circulation, but diminished concomitant with activation of CD4+ T and B cells. ELISPOT assays documented robust responses to hF.IX in the draining lymph nodes of injected muscle by day 14. Formation of inhibitory antibodies to hF.IX was observed over a wide range of vector doses, with increased doses causing stronger immune responses. A prolonged inflammatory reaction in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene expression. By 1.5 months, hF.IX antigen re-emerged in circulation in approximately 70% of animals injected with high vector dose. Hepatic gene transfer elicited only infrequent and weaker immune responses, with higher vector doses causing a reduction in T-cell responses to hF.IX. In summary, the data document substantial influence of target tissue, local antigen presentation, and antigen levels on lymphocyte responses to F.IX.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  19. Highly stable beta-class carbonic anhydrases useful in carbon capture systems

    DOEpatents

    Alvizo, Oscar; Benoit, Mike; Novick, Scott

    2013-04-16

    The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

  20. Highly stable beta-class carbonic anhydrases useful in carbon capture systems

    DOEpatents

    Alvizo, Oscar; Benoit, Michael R; Novick, Scott J

    2013-08-20

    The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

  1. Lead Development of Thiazolylsulfonamides with Carbonic Anhydrase Inhibitory Action.

    PubMed

    Carta, Fabrizio; Birkmann, Alexander; Pfaff, Tamara; Buschmann, Helmut; Schwab, Wilfried; Zimmermann, Holger; Maresca, Alfonso; Supuran, Claudiu T

    2017-03-17

    A series of congeners structurally related to pritelivir, N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide, a helicase-primase inhibitor for the treatment of herpes simplex virus infections, was prepared. The synthesized primary and secondary sulfonamides were investigated as inhibitors of six physiologically and pharmacologically relevant human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic enzymes hCA I and II, the mitochondrial ones hCA VA and VB, and the transmembrane, tumor associated hCA IX and XII. Low nanomolar inhibition KI values were detected for all of them, with a very interesting and well-defined structure-activity relationship. As many CAs are involved in serious pathologies, among which are cancer, obesity, epilepsy, glaucoma, etc., sulfonamide inhibitors as those reported here may be of interest as drug candidates. Furthermore, pritelivir itself is an effective inhibitor of some CAs, also inhibiting whole blood enzymes from several mammalian species, which may be a favorable pharmacokinetic feature of the drug which can be transported throughout the body bound to blood CA I and II.

  2. Muscle as a target for supplementary factor IX gene transfer.

    PubMed

    Hoffman, Brad E; Dobrzynski, Eric; Wang, Lixin; Hirao, Lauren; Mingozzi, Federico; Cao, Ou; Herzog, Roland W

    2007-07-01

    Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.

  3. Title IX: Boom or Bust?

    ERIC Educational Resources Information Center

    Mather, Marilyn J.

    2003-01-01

    Athletics has been significantly impacted by Title IX through an increase the number of female athletes, the number of teams available, and indirectly, the development of women's professional leagues. However, women in leadership positions in athletics have declined significantly since Title IX was signed into law. A concern about the…

  4. Carbonic anhydrase inhibitors drug design.

    PubMed

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

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

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

    PubMed

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

    2016-06-23

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

  6. Synthesis and carbonic anhydrase inhibition of a series of SLC-0111 analogs.

    PubMed

    Carta, Fabrizio; Vullo, Daniela; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T

    2017-05-01

    SLC-0111 is a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor (CAI) in Phase I/II clinical trials for the treatment of advanced hypoxic tumors complicated with metastases. Its antitumor effects are due to inhibition of the enzymatic activity of CA IX, an isoform predominantly found in tumors/metastases, but it also reduces the cancer stem cells population. Here we report the synthesis of analogs of SLC-0111, both of the sulfanilamide and metanilamide series, which possess diverse substitution patterns at the terminal ureido-phenyl moiety, thus including one or more halogens, trifluoromethyl, perchloro-/perfluorophenyl groups instead of the 4-fluorophenyl present in SLC-0111. Most of the sulfanilamide ureido derivatives were highly effective inhibitors of the tumor associated isoform and some showed selective CA IX/XII inhibitory profiles. Most of the sulfanilamide ureido derivatives were highly effective and in some cases selective CA IX/XII inhibitors, whereas the metanilamide ureido derivatives were less effective as transmembrane CA isoforms inhibitors. Structure activity relationship for this class of sulfonamides is discussed in detail.

  7. Thermostable Carbonic Anhydrases in Biotechnological Applications

    PubMed Central

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

    2015-01-01

    Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application. PMID:26184158

  8. Oxygen Availability for Porphyrin Biosynthesis Enzymes Determines the Production of Protoporphyrin IX (PpIX) during Hypoxia

    PubMed Central

    Otsuka, Shimpei; Matsumoto, Kentaro; Nakajima, Motowo; Tanaka, Tohru; Ogura, Shun-ichiro

    2015-01-01

    5-Aminolevulinic acid (ALA), a precursor of porphyrin, is specifically converted to the fluorescent substance protoporphyrin IX (PpIX) in tumors to be used as a prodrug for photodynamic therapy and diagnosis. Hypoxia, a common feature of solid tumors, decreases the efficacy of ALA-based photodynamic therapy and diagnosis. This decrease results from the excretion of porphyrin precursor coproporphyrinogen III (CPgenIII), an intermediate in the biosynthesis of PpIX. However, the mechanism of CPgenIII excretion during hypoxia remains unclear. In this study, we revealed the importance of mitochondrial respiration for the production of PpIX during hypoxia. Porphyrin concentrations were estimated in human gastric cancer cell lines by HPLC. Expression levels of porphyrin biosynthesis genes were measured by qRT-PCR and immunoblotting. Blockage of porphyrin biosynthesis was an oxygen-dependent phenomenon resulting from decreased PpIX production in mitochondria under hypoxic conditions. PpIX production was increased by the inhibition of mitochondrial respiration complexes, which indicates that the enzymes of porphyrin biosynthesis compete with respiration complexes for molecular oxygen. Our results indicate that targeting the respiration complexes is a rationale for enhancing the effect of ALA-mediated treatment and diagnosis. PMID:26717566

  9. Anti-inflammatory and carbonic anhydrase restoring actions of yam powder (Dioscorea spp) contribute to the prevention of cysteamine-induced duodenal ulcer in a rat model.

    PubMed

    Park, Jong-Min; Kim, Yoon-Jae; Kim, Ju-Seung; Han, Young-Min; Kangwan, Napapan; Hahm, Ki Baik; Kim, Tae-Sok; Kwon, Oran; Kim, Eun-Hee

    2013-08-01

    Increased acid output, accompanied with a defective defense system, is considered a fundamental pathogenesis of duodenal ulcer (DU). However, relapse of DU occurs despite proton pump inhibitors and H2 receptor antagonists, hence imposing the enforcement of the defense system. Dried powder of the yam tuber (Dioscorea spp) has been used in traditional folk medicine as a nutritional fortification. We hypothesized that dried-yam powder would prevent DU through improvement of anti-inflammatory actions and carbonic anhydrase (CA) activity. Therefore, we investigated the preventive effects of dried-yam powder against the cysteamine-induced DU and elucidated the underlying mechanisms. Duodenal ulcers were induced in Sprague-Dawley rats by intragastric administration of 500 mg/kg cysteamine-HCl. The dried-yam powder was used as a pretreatment before the cysteamine-HCl. The number and size of DU were measured. The expressions of inflammation mediators were checked in duodenal tissues, and the expressions of CAs and malondialdehyde levels were also examined. Cysteamine provoked perforated DU, whereas dried-yam powder significantly prevented DU as much as pantoprazole and significantly reduced the incidence of perforation. The messenger RNA expressions of cyclooxygenase-2 and inducible nitric oxide synthase were remarkably decreased in the yam group compared with the cysteamine group, and the serum levels of proinflammatory cytokines including interleukin-1β, interleukin-6, and tumor necrosis factor were significantly attenuated in the yam group. Cysteamine significantly decreased the expression of CAs, whereas yam treatment significantly preserved the expressions of CA IX, XII, and XIV. In conclusion, dried-yam powder exerts a significant protective effect against cysteamine-induced DU by lowering the activity of inflammatory cytokines and free radicals and restoring the activity of CAs, except in CA IV.

  10. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques specific carbonic anhydrase protein molecules in serum and other body fluids. Measurements of carbonic anhydrase B and C aid in the diagnosis of abnormal hemoglobin metabolism....

  11. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques specific carbonic anhydrase protein molecules in serum and other body fluids. Measurements of carbonic anhydrase B and C aid in the diagnosis of abnormal hemoglobin metabolism....

  12. Physiological functions of the alpha class of carbonic anhydrases.

    PubMed

    Frost, Susan C

    2014-01-01

    Carbonic anhydrases are ubiquitous enzymes that catalyze the reversible hydration of carbon dioxide. These enzymes are of ancient origin as they are found in the deepest of branches of the evolutionary tree. Of the five different classes of carbonic anhydrases, the alpha class has perhaps received the most attention because of its role in human pathology. This review focuses on the physiological function of this class of carbonic anhydrases organized by their cellular location.

  13. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes.

    PubMed Central

    Goldring, M B; Birkhead, J; Sandell, L J; Kimura, T; Krane, S M

    1988-01-01

    In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions. Images PMID:3264290

  14. Mg-Protoporphyrin IX Signals Enhance Plant's Tolerance to Cold Stress.

    PubMed

    Zhang, Zhong-Wei; Wu, Zi-Li; Feng, Ling-Yang; Dong, Li-Hua; Song, An-Jun; Yuan, Ming; Chen, Yang-Er; Zeng, Jian; Chen, Guang-Deng; Yuan, Shu

    2016-01-01

    The relationship between Mg-protoporphyrin IX (Mg-Proto IX) signals and plant's tolerance to cold stress is investigated. Arabidopsis seedlings grown for 3 weeks were pretreated with 2 mM glutamate (Glu) and 2 mM MgCl2 for 48 h at room temperature to induce Mg-Proto IX accumulation. Then cold stress was performed at 4°C for additional 72 h. Glu + MgCl2 pre-treatments alleviated the subsequent cold stress significantly by rising the leaf temperature through inducing Mg-Proto IX signals. The protective role of Glu + MgCl2 treatment was greatly compromised in the mutants of Mg-Proto IX synthesis, Mg-Proto IX signaling, and cyanide-resistant respiration. And the enhancement of cold-responsive gene expression was greatly compromised in the mutants of Mg-Proto IX synthesis, Mg-Proto IX signaling and ABA signaling, but not in the mutant of cyanide-resistant respiration. Cold stress promoted cyanide-resistant respiration and leaf total respiration exponentially, which could be further induced by the Glu + MgCl2 treatment. Mg-Proto IX signals also activate antioxidant enzymes and increase non-enzymatic antioxidants [glutathione but not ascorbic acid (AsA)] to maintain redox equilibrium during the cold stress.

  15. Mg-Protoporphyrin IX Signals Enhance Plant’s Tolerance to Cold Stress

    PubMed Central

    Zhang, Zhong-Wei; Wu, Zi-Li; Feng, Ling-Yang; Dong, Li-Hua; Song, An-Jun; Yuan, Ming; Chen, Yang-Er; Zeng, Jian; Chen, Guang-Deng; Yuan, Shu

    2016-01-01

    The relationship between Mg-protoporphyrin IX (Mg-Proto IX) signals and plant’s tolerance to cold stress is investigated. Arabidopsis seedlings grown for 3 weeks were pretreated with 2 mM glutamate (Glu) and 2 mM MgCl2 for 48 h at room temperature to induce Mg-Proto IX accumulation. Then cold stress was performed at 4°C for additional 72 h. Glu + MgCl2 pre-treatments alleviated the subsequent cold stress significantly by rising the leaf temperature through inducing Mg-Proto IX signals. The protective role of Glu + MgCl2 treatment was greatly compromised in the mutants of Mg-Proto IX synthesis, Mg-Proto IX signaling, and cyanide-resistant respiration. And the enhancement of cold-responsive gene expression was greatly compromised in the mutants of Mg-Proto IX synthesis, Mg-Proto IX signaling and ABA signaling, but not in the mutant of cyanide-resistant respiration. Cold stress promoted cyanide-resistant respiration and leaf total respiration exponentially, which could be further induced by the Glu + MgCl2 treatment. Mg-Proto IX signals also activate antioxidant enzymes and increase non-enzymatic antioxidants [glutathione but not ascorbic acid (AsA)] to maintain redox equilibrium during the cold stress. PMID:27803706

  16. Non-Classical Inhibition of Carbonic Anhydrase

    PubMed Central

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

    2016-01-01

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

  17. Overview of the carbonic anhydrase family.

    PubMed

    McKenna, Robert; Frost, Susan C

    2014-01-01

    The purpose of this collection of chapters is to provide a glimpse of where the carbonic anhydrase (CA) field is. This book is by no means fully inclusive, as only a few of the lead researchers around the world contributed; it serves only to show that the CA field is still pushing the boundaries of research as it has done since its discovery, and will do for a long time to come.

  18. Localization of type II collagen, long form alpha 1(IX) collagen, and short form alpha 1(IX) collagen transcripts in the developing chick notochord and axial skeleton.

    PubMed

    Swiderski, R E; Solursh, M

    1992-06-01

    In this study we compare, by in situ hybridization, the spatial and temporal expression patterns of transcripts of avian type II collagen and the long and short forms of the (alpha 1) chain of type IX collagen during the development of the notochord and axial skeleton. We observed type II collagen and short form type IX collagen transcripts in the developing (stage 25-28) nonchondrogenic notochord. Conversely, long form type IX transcripts were not detectable in the notochord or perinotochordal sheath. Interestingly, all three transcripts colocalized in the developing chondrogenic vertebrae of the axial skeleton as well as in the chondrocranium and Meckel's cartilage. The expression of the short form of type IX collagen in these regions was more restricted than that of the long form. This report provides additional support for a complex regulatory pathway of cartilage marker gene expression in chondrogenic vs. nonchondrogenic tissues during avian embryogenesis.

  19. Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine.

    PubMed

    Aksu, Kadir; Nar, Meryem; Tanc, Muhammet; Vullo, Daniela; Gülçin, Ilhami; Göksu, Süleyman; Tümer, Ferhan; Supuran, Claudiu T

    2013-06-01

    A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K(i)s were in the range of 0.061-1.822 μM for CA I, 1.47-2.94 nM for CA II, 2.25-3.34 μM for CA VA, 0.041-0.37 μM for CA IX, 0.021-1.52 μM for CA XII, 0.007-0.219 μM for CA XIV, 0.35-5.31 μM for CgCA and 0.465-4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.

  20. Reconsidering the Status of Title IX.

    ERIC Educational Resources Information Center

    Hammer, Ben

    2003-01-01

    Discusses the controversy over Title IX and women's participation in college athletics. Critics say the mandate shortchanges men's teams, while proponents say that women's sports programs remain underfunded in spite of Title IX. Describes some proposed modifications to Title IX and their potential effects. (SLD)

  1. The product of the adenovirus intermediate gene IX is a transcriptional activator.

    PubMed Central

    Lutz, P; Rosa-Calatrava, M; Kedinger, C

    1997-01-01

    We have investigated the functional properties of the product of the adenovirus type 5 gene IX. This gene, which is expressed at intermediate times postinfection, encodes a small polypeptide (pIX) of 140 residues that has previously been shown to be incorporated into the viral capsid. Here, we show that pIX, in addition to its structural contribution, exhibits transcriptional properties. In transient transfection experiments, expression of pIX stimulated adenovirus major late promoter activity. The effect was independent of other viral proteins, but the level of promoter activation appeared strongly pIX dose dependent; similar levels of induction were observed with other cellular or viral TATA-containing (but not with TATA-less) promoters. This promoter specificity could be reproduced in a cell-free transcription system by the addition of purified recombinant pIX, further stressing the transcriptional nature of the phenomenon. A preliminary structural analysis of pIX indicated that the integrity of a putative leucine zipper at the carboxy-terminal end of the molecule, as well as elements within the amino-terminal half, was critical for pIX transcriptional activity. The relevance of these findings in adenovirus infection is discussed. PMID:9188576

  2. [Title IX and Intercollegiate Athletics.

    ERIC Educational Resources Information Center

    Department of Health , Education, and Welfare, Washington., DC. Office of the Secretary.

    Title IX of the 1972 Education Amendments is the topic of these Department of Health, Education, and Welfare (DHEW) notices, which include both a proposed policy interpretation regarding intercollegiate athletic programs and a proposed amendment regarding federal regulation of school dress codes. The purpose of the first action is to ensure that…

  3. Radioimmunoassay of carbonic anhydrase III in rat tissues.

    PubMed Central

    Shiels, A; Jeffery, S; Wilson, C; Carter, N

    1984-01-01

    A specific and sensitive radioimmunoassay for the rat carbonic anhydrase III isoenzyme was developed. High concentrations of carbonic anhydrase III were detected in soleus muscle and male liver. Female liver and other skeletal muscles contained significantly lower concentrations, and only trace amounts were found in heart, prostate, kidney, brain, plasma, urine and, possibly, erythrocytes. PMID:6424658

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

    PubMed

    Rasmussen, Jacob K; Boedtkjer, Ebbe

    2017-01-01

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

  5. Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6-dihydropyrimidine-benzenesulfonamides acting as carbonic anhydrase inhibitors.

    PubMed

    Vullo, Daniela; Supuran, Claudiu T; Scozzafava, Andrea; De Simone, Giuseppina; Monti, Simona Maria; Alterio, Vincenzo; Carta, Fabrizio

    2016-08-15

    Herein we report an in vitro kinetic evaluation against the most relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms (I, II, IX and XII) of a small series of lactate dehydrogenase (LDH, EC 1.1.1.27) inhibitors. All compounds contain a primary sulfonamide zinc-binding group (ZBG) substituted with the 2-thio-6-oxo-1,6-dihydropyrimidine scaffold. By means of X-ray crystallographic experiments we explored the ligand-enzyme binding modes, thus highlighting the contribution of the 2-thio-6-oxo-1,6-dihydropyrimidine moiety to the stabilization of the complex.

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

    PubMed

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

    2016-01-15

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

  7. First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus.

    PubMed

    Jeong, Ji-Min; Kim, Ju-Won; Kim, Do-Hyung; Park, Chan-Il

    2015-04-01

    CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

  8. Paralysis Episodes in Carbonic Anhydrase II Deficiency.

    PubMed

    Al-Ibrahim, Alia; Al-Harbi, Mosa; Al-Musallam, Sulaiman

    2003-01-01

    Carbonic anhydrase II (CAII) deficiency is an autosomal recessive disorder manifest by osteopetrosis, renal tubular acidosis, and cerebral calcification. Other features include growth failure and mental retardation. Complications of the osteopetrosis include frequent bone fractures, cranial nerve compression, and dental mal-occlusion. A hyper-chloremic metabolic acidosis, sometimes with hypokalemia, occurs due to renal tubular acidosis that may be proximal, distal, or more commonly, the combined type. Such patients may present with global hypotonia, muscle weakness or paralysis. We report a case of CA II deficiency with recurrent attacks of acute paralysis which was misdiagnosed initially as Guillian-Barre syndrome.

  9. Methods of producing protoporphyrin IX and bacterial mutants therefor

    SciTech Connect

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  10. Role of the HBx oncoprotein in carbonic anhydrase 9 induction.

    PubMed

    Holotnakova, Tereza; Tylkova, Lucia; Takacova, Martina; Kopacek, Juraj; Petrik, Juraj; Pastorekova, Silvia; Pastorek, Jaromir

    2010-01-01

    Carbonic anhydrase 9 (CA9), as one of the most hypoxia-responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia-inducible factor 1 (HIF-1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF-1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT-PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF-1 and a functional HRE element located -10/-3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis.

  11. Chemically modified carbonic anhydrases useful in carbon capture systems

    DOEpatents

    Novick, Scott J; Alvizo, Oscar

    2013-10-29

    The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

  12. Chemically modified carbonic anhydrases useful in carbon capture systems

    DOEpatents

    Novick, Scott; Alvizo, Oscar

    2013-01-15

    The present disclosure relates to chemically modified carbonic anhydrase polypeptides and soluble compositions, homogenous liquid formulations comprising them. The chemically modified carbonic anhydrase polypeptides have improved properties relative to the same carbonic anhydrase polypeptide that is not chemically modified including the improved properties of increased activity and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides methods of preparing the chemically modified polypeptides and methods of using the chemically modified polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering.

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

    PubMed

    Hu, Honghong; Boisson-Dernier, Aurélien; Israelsson-Nordström, Maria; Böhmer, Maik; Xue, Shaowu; Ries, Amber; Godoski, Jan; Kuhn, Josef M; Schroeder, Julian I

    2010-01-01

    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.

  14. Mechanisms of Vesicular Stomatitis Virus Inactivation by Protoporphyrin IX, Zinc-protoporphyrin IX and Mesoporphyrin IX.

    PubMed

    Cruz-Oliveira, Christine; Almeida, Andreza F; Freire, João M; Caruso, Marjolly B; Morando, Maria A; Ferreira, Vivian N S; Assunção-Miranda, Iranaia; Gomes, Andre M O; Castanho, Miguel A R B; Da Poian, Andrea T

    2017-03-27

    Virus resistance to antiviral therapies is an increasing concern that makes urgent the development of broad spectrum antiviral drugs. Targeting viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins, due to their relative hydrophobicity and pro-oxidant character, are good candidates for antivirals' development. In the present work, we characterized the antiviral activity of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX) and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV), and evaluated the mechanisms involved in this activity. VSV treatment with PPIX, ZnPPIX and MPIX promoted a dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV inactivation by porphyrins, suggesting that singlet oxygen ((1)O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, (1)O2 was detected by DMA oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX and MPIX as good models for broad antiviral drug design.

  15. Expression and Prognostic Significance of a Panel of Tissue Hypoxia Markers in Head-and-Neck Squamous Cell Carcinomas

    SciTech Connect

    Le, Quynh-Thu Kong, Christina; Lavori, Phillip W.; O'Byrne, Ken; Erler, Janine T.; Huang Xin; Chen Yijun; Cao Hongbin; Tibshirani, Robert; Denko, Nic; Giaccia, Amato J.; Koong, Albert C.

    2007-09-01

    Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO{sub 2} and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, I{kappa}B kinase {beta}, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO{sub 2} measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO{sub 2}, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO{sub 2} (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy.

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

    PubMed Central

    Belkaid, Anissa; Čuperlović-Culf, Miroslava; Touaibia, Mohamed; Ouellette, Rodney J.; Surette, Marc E.

    2016-01-01

    Metabolic shift is one of the major hallmarks of cancer development. Estrogen receptor (ER) activity has a profound effect on breast cancer cell growth through a number of metabolic changes driven by its effect on transcription of several enzymes, including carbonic anhydrases, Stearoyl-CoA desaturase-1, and oncogenes including HER2. Thus, estrogen receptor activators can be expected to lead to the modulation of cell metabolism in estrogen receptor positive cells. In this work we have investigated the effect of 17β-estradiol, an ER activator, and ferulic acid, a carbonic anhydrase inhibitor, as well as ER activator, in the absence and in the presence of the carbonic anhydrase inhibitor acetazolamide on the metabolism of MCF7 cells and MCF7 cells, stably transfected to express HER2 (MCF7HER2). Metabolic profiles were studied using 1D and 2D metabolomic Nuclear Magnetic Resonance (NMR) experiments, combined with the identification and quantification of metabolites, and the annotation of the results in the context of biochemical pathways. Overall changes in hydrophilic metabolites were largest following treatment of MCF7 and MC7HER2 cells with 17β-estradiol. However, the carbonic anhydrase inhibitor acetazolamide had the largest effect on the profile of lipophilic metabolites. PMID:27240414

  17. 34 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2016-07-01

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  18. 34 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2015-07-01

    ... 34 Education 1 2015-07-01 2015-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of the...] Procedures. Subject Index Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph...

  19. 45 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2006-10-01

    ... 45 Public Welfare 1 2006-10-01 2006-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTHAND... Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are in brackets [ ]....

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  2. 45 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2016-10-01

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  3. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2002-10-01

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  4. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2008-10-01

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  5. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2007-07-01

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  6. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation \\1\\

    Code of Federal Regulations, 2010 CFR

    1997-10-01

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  7. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2005-07-01

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  8. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2009-07-01

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  9. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2004-07-01

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  10. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2008-07-01

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  11. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation \\1\\

    Code of Federal Regulations, 2010 CFR

    1996-10-01

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  12. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2003-10-01

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  13. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2002-07-01

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  14. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2001-10-01

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  15. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2006-07-01

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  16. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2007-10-01

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  17. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2005-10-01

    ... 45 Public Welfare 1 2005-10-01 2005-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH AND... to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are in brackets . A Access...

  18. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation \\1\\

    Code of Federal Regulations, 2010 CFR

    1999-10-01

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  19. 45 CFR Subject Index to Title IX... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2015-10-01

    ... 45 Public Welfare 1 2015-10-01 2015-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare Department of Health and... Subject Index to Title IX Preamble and Regulation 1 1 Preamble paragraph numbers are in brackets [ ]....

  20. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2004-10-01

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  1. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2003-07-01

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  2. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2009-10-01

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  3. Carbonic Anhydrase Catalysis: An Experiment on Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Spyridis, Greg T.; And Others

    1985-01-01

    Describes an undergraduate enzyme kinetics experiment which uses bovine erythrocyte carbonic anhydrase, a very stable enzyme commercially available in lyophilized form. Includes background information, reactions involved, procedures used, and the calculation of typical results obtained. (JN)

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

    SciTech Connect

    Mitterberger, Maria C.; Kim, Geumsoo; Rostek, Ursula; Levine, Rodney L.; Zwerschke, Werner

    2012-05-01

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

  5. Title IX and Sex Discrimination. Revised.

    ERIC Educational Resources Information Center

    Office for Civil Rights (ED), Washington, DC.

    Title IX of the Education Amendments of 1972 protects people from discrimination based on sex in education programs or activities that receive Federal financial assistance. This brochure outlines the responsibilities of education programs and activities covered by Title IX, the responsibilities of the Office for Civil Rights (OCR) in enforcing…

  6. Title IX and Home Economics Instruction

    ERIC Educational Resources Information Center

    Thurston, Paul W.

    1977-01-01

    The following are discussed with reference to title IX of the Education Amendments of 1972, which prohibit sex discrimination in federally-supported educational programs: What is title IX? The letter of the law--can the school limit home economics to girls? Is a school in noncompliance if over 75 percent of the students in a home economics class…

  7. Statistical Evidence and Compliance with Title IX

    ERIC Educational Resources Information Center

    Cheslock, John J.; Eckes, Suzanne E.

    2008-01-01

    The scope of Title IX clearly includes all aspects of education, but the legislation's application to college athletics receives the most attention. Athletics programs, unlike most academic activities, are sex segregated, so the proper interpretation of the intercollegiate athletics provisions of Title IX is less clear-cut. This article examines…

  8. Intersex (ix) mutations of Drosophila melanogaster cause nonrandom cell death in genital disc and can induce tumours in genitals in response to decapentaplegic (dpp(disk)) mutations.

    PubMed

    Chatterjee, R N; Chatterjee, P; Kuthe, S; Acharyya-Ari, M; Chatterjee, R

    2015-06-01

    In Drosophila melanogaster, the intersex (ix) is a terminally positioned gene in somatic sex determination hierarchy and function with the female specific product of double sex (DSX(F)) to implement female sexual differentiation. The null phenotype of ix is to transform diplo-X individuals into intersexes while leaving haplo-X animals unaffected. This study on the effect of different intersex mutations on genital disc development provides the following major results: (i) similar range of a characteristic array of morphological structures (from almost double sex terminalia to extreme reduction of terminal appendages) was displayed by the terminalia of XX ix(1)/ix(1) , XX ix(2)/ix(2) and XX ix(5)/ix(5) individuals; (ii) an increased number of apoptotic cells were found to occur in a localized manner in mature third instar larval genital discs of ix individuals; (iii) ix mutations can induce high frequency of neoplastic tumours in genitals in the presence of decapentaplegic (dpp(disk)) mutations; and (iv) heteroallelic combinations of dpp(disk) mutations can also induce tumours in intersex genitals with variable expressivity. On the basis of these findings, we suggest that: (i) loss of function of ix causes massive cell death in both male and female genital primordia of genital discs, resulting phenotype mimicking in male and female characteristics in genitals; and (ii) at the discs, the apoptotic cells persist as 'undead' cells that can induce oncogenic transformation in the neighbouring disc cells when dpp signalling is blocked or reduced by dpp(disk) mutations.

  9. New approach of delivering cytotoxic drugs towards CAIX expressing cells: A concept of dual-target drugs.

    PubMed

    van Kuijk, Simon J A; Parvathaneni, Nanda Kumar; Niemans, Raymon; van Gisbergen, Marike W; Carta, Fabrizio; Vullo, Daniela; Pastorekova, Silvia; Yaromina, Ala; Supuran, Claudiu T; Dubois, Ludwig J; Winum, Jean-Yves; Lambin, Philippe

    2017-02-15

    Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies.

  10. Carbon dioxide "trapped" in a β-carbonic anhydrase

    DOE PAGES

    Aggarwal, Mayank; Chua, Teck Khiang; Pinard, Melissa A.; ...

    2015-10-12

    Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/ dehydration of CO2/HCO3- with rates approaching diffusion-controlled limits (kcat/KM ~ 108 M–1s–1). Here, this family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a beta-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryocooled crystallography. The structure has been refined to 1.6 angstrom resolution with Rcryst and Rfree values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to havemore » CO2, captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2, binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2, binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dirners or higher-order oligomeric states, and the CO2, bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.« less

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

    ERIC Educational Resources Information Center

    McQuate, Robert S.; Reardon, John E.

    1978-01-01

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

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

    PubMed

    Becker, Holger M; Deitmer, Joachim W

    2007-05-04

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

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

    PubMed

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

    2017-01-20

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

  14. A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing.

    PubMed

    Lecoeur, Marie; Goossens, Jean-François; Vaccher, Claude; Bonte, Jean-Paul; Foulon, Catherine

    2011-10-01

    Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico-chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV-detectable pI markers using a central composite design. The separation was performed in a fused-silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol-water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.

  15. A Class of 4-Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective Carbonic Anhydrase Inhibitory Action and Antiglaucoma Effects

    PubMed Central

    2015-01-01

    We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure–activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a–c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII. PMID:25358036

  16. Multimeric xanthates as carbonic anhydrase inhibitors.

    PubMed

    Abellán-Flos, Marta; Tanç, Muhammet; Supuran, Claudiu T; Vincent, Stéphane P

    2016-12-01

    The field of multivalent inhibition of enzymes is growing exponentially from the first reported multivalent effect on a glycosidase enzyme. However, the investigations have generally remained restricted to carbohydrate-processing enzymes. Carbonic anhydrases are ubiquitous metallo-enzymes involved in many key biological processes, that catalyze the reversible hydration/dehydration of [Formula: see text]. This study reports the first synthesis of multimeric xanthates addressing the selectivity and potency of CA multivalent inhibition. Six multivalent compounds containing three, four, and six xanthate moieties were prepared and assayed against four relevant CA isoforms together with their monovalent analogues. Some of the multimers were stronger inhibitors than the monomeric species. For hCA I, the two best molecules 18 and 20 showed an improvement of the ligand affinity of 4.8 and 2.3 per xanthate units (valence-corrected values), respectively, which corresponds to a clear multivalent effect. Moreover, the biochemical assays demonstrated that the multimeric presentation of xanthates, also affected the selectivity of the relative inhibition among the four CAs assayed.

  17. Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency)

    SciTech Connect

    Taylor, S.A.M.; Deugau, K.V.; Lillicrap, D.P. )

    1991-01-01

    Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which {gt}95% are single nulceotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. The authors used differential termination of primer extension to confirm and measure the degree of mosaicism. The study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male.

  18. Functional Analysis of Adenovirus Protein IX Identifies Domains Involved in Capsid Stability, Transcriptional Activity, and Nuclear Reorganization

    PubMed Central

    Rosa-Calatrava, Manuel; Grave, Linda; Puvion-Dutilleul, Francine; Chatton, Bruno; Kedinger, Claude

    2001-01-01

    The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology. PMID:11435594

  19. A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

    SciTech Connect

    Kim, Jung D.; Seo, Kwang W.; Lee, Eun A.; Quang, Nguyen N.; Cho, Hong R.; Kwon, Byungsuk

    2011-07-01

    Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

  20. Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

    PubMed Central

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

    2016-01-01

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

  1. Spectral action for Bianchi type-IX cosmological models

    NASA Astrophysics Data System (ADS)

    Fan, Wentao; Fathizadeh, Farzad; Marcolli, Matilde

    2015-10-01

    A rationality result previously proved for Robertson-Walker metrics is extended to a homogeneous anisotropic cosmological model, namely the Bianchi type-IX minisuperspace. It is shown that the Seeley-de Witt coefficients appearing in the expansion of the spectral action for the Bianchi type-IX geometry are expressed in terms of polynomials with rational coefficients in the cosmic evolution factors w 1( t) , w 2( t) , w 3( t) , and their higher derivates with respect to time. We begin with the computation of the Dirac operator of this geometry and calculate the coefficients a 0 ,a 2 ,a 4 of the spectral action by using heat kernel methods and parametric pseudodifferential calculus. An efficient method is devised for computing the Seeley-de Witt coefficients of a geometry by making use of Wodzicki's noncommutative residue, and it is confirmed that the method checks out for the cosmological model studied in this article. The advantages of the new method are discussed, which combined with symmetries of the Bianchi type-IX metric, yield an elegant proof of the rationality result.

  2. Platelet Glycoprotein Ib-IX and Malignancy

    DTIC Science & Technology

    2010-09-01

    cancer to metastatic disease . During the next year we propose to examine the relevance of platelet receptors in models of spontaneous metastasis. A...the prognosis for recovery from breast cancer cannot be under emphasized. Indeed, the spread of metastatic disease represents a fundamental change in...IX have been identified, including von Willebrand factor (vWF) and thrombin, illustrating platelet GP Ib-IX as a major initiator of platelet thrombus

  3. Carbonic anhydrases in chick extra-embryonic structures: a role for CA in bicarbonate reabsorption through the chorioallantoic membrane.

    PubMed

    Gabrielli, M Gabriella

    2004-06-01

    The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and HCO3(-)/Cl(-) anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.

  4. Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

    PubMed Central

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

    2013-01-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

  5. Characteristics of recombinant α-carbonic anhydrase of polyextremophilic bacterium Bacillus halodurans TSLV1.

    PubMed

    Faridi, Shazia; Satyanarayana, T

    2016-08-01

    Carbonic anhydrase (CA) is a biocatalyst that catalyzes the hydration of CO2 to bicarbonate and protons, thus useful in mitigating green house effect by sequestering CO2 from various point sources. An alkalistable and moderately thermostable α- carbonic anhydrase encoding gene (BhCA) from Bacillus halodurans TSLV1 has been cloned and expressed in Escherichia coli. A 31.4-fold enhancement in CA production was achieved due to cloning and expression in E. coli. About 50% of the CA produced was secreted when recombinant E. coli with BhCA-pET22b was cultivated in a medium with EDTA and lysozyme because of the efficient pelB leader sequence. rBhCA is a ∼75kDa homodimeric protein with a Tm of 72°C and T1/2 values of 66 and 24min at 50 and 60°C, respectively. SDM analysis revealed that H137, H139, H156 and H110 present in the active site play an important role in catalysis. Mineralization of CO2 using rBhCA led to the accelerated precipitation of CaCO3 in calcite form. rBhCA also functions as an efficient virtual peroxidase when Zn(2+) is substituted with Mn(2+).

  6. Regulation of tumor pH and the role of carbonic anhydrase 9.

    PubMed

    Swietach, Pawel; Vaughan-Jones, Richard D; Harris, Adrian L

    2007-06-01

    The high metabolic rate required for tumor growth often leads to hypoxia in poorly-perfused regions. Hypoxia activates a complex gene expression program, mediated by hypoxia inducible factor 1 (HIF1alpha). One of the consequences of HIF1alpha activation is up-regulation of glycolysis and hence the production of lactic acid. In addition to the lactic acid-output, intracellular titration of acid with bicarbonate and the engagement of the pentose phosphate shunt release CO(2) from cells. Expression of the enzyme carbonic anhydrase 9 on the tumor cell surface catalyses the extracellular trapping of acid by hydrating cell-generated CO(2) into [see text] and H(+). These mechanisms contribute towards an acidic extracellular milieu favoring tumor growth, invasion and development. The lactic acid released by tumor cells is further metabolized by the tumor stroma. Low extracellular pH may adversely affect the intracellular milieu, possibly triggering apoptosis. Therefore, primary and secondary active transporters operate in the tumor cell membrane to protect the cytosol from acidosis. We review mechanisms regulating tumor intracellular and extracellular pH, with a focus on carbonic anhydrase 9. We also review recent evidence that may suggest a role for CA9 in coordinating pH(i) among cells of large, unvascularized cell-clusters.

  7. Engineered Escherichia coli with periplasmic carbonic anhydrase as a biocatalyst for CO2 sequestration.

    PubMed

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun; Cha, Hyung Joon

    2013-11-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

  8. Association between FBP1 and hypoxia-related gene expression in clear cell renal cell carcinoma

    PubMed Central

    NING, XIANG-HUI; LI, TENG; GONG, YAN-QING; HE, QUN; SHEN, QI; PENG, SHUANG-HE; WANG, JIANG-YI; CHEN, JIN-CHAO; GUO, YING-LU; GONG, KAN

    2016-01-01

    Fructose-1,6-bisphosphatase 1 (FBP1) is a rate-limiting enzyme in gluconeogenesis. Recently, the catalytic activity-independent function of FBP1, hypoxia-induced factor (HIF) repression in the nucleus, was identified. The aim of the present study was to investigate the association between FBP1 and hypoxia-related gene expression in clear cell renal cell carcinoma (ccRCC). The protein expression levels of FBP1, HIF-1α, HIF-2α, erythropoietin (EPO) and carbonic anhydrase IX (CA9) were assessed by immunohistochemical staining of ccRCC paraffin blocks from 123 patients using the tissue microarray technique. The expression level of FBP1 was then correlated with various clinicopathological factors, and the protein expression levels of HIF-1α, HIF-2α, EPO and CA9. Clinicopathological factors, including age, gender, T stage and Fuhrman grade, were not significantly different between patients with low and high FBP1 expression in ccRCC (P>0.05). FBP1 protein expression level was significantly correlated with the expression levels of HIF-1α (P=0.005) and EPO (P=0.010), but not significantly correlated with the expression levels of HIF-2α (P=0.123) and CA9 (P=0.513) in ccRCC tissues. The current findings confirm the association between FBP1 and hypoxia-related gene expression, and may facilitate understanding of the mechanisms of ccRCC tumorigenesis. PMID:27313747

  9. Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics.

    PubMed

    Schroeder, Marie A; Ali, Mohammad A; Hulikova, Alzbeta; Supuran, Claudiu T; Clarke, Kieran; Vaughan-Jones, Richard D; Tyler, Damian J; Swietach, Pawel

    2013-03-05

    CO2 is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2 clearance by catalyzing CO2 hydration (to H(+) and HCO3(-)), thereby changing the gradient for CO2 venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO2, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by (13)C magnetic resonance spectroscopy from the rate of H(13)CO3(-) production from (13)CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-(13)C]pyruvate. CA activity measured upon [1-(13)C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by (31)P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2 venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart's energetic

  10. Carbon dioxide "trapped" in a β-carbonic anhydrase

    SciTech Connect

    Aggarwal, Mayank; Chua, Teck Khiang; Pinard, Melissa A.; Szebenyi, Doletha M.; McKenna, Robert

    2015-10-12

    Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/ dehydration of CO2/HCO3- with rates approaching diffusion-controlled limits (kcat/KM ~ 108 M–1s–1). Here, this family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a beta-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryocooled crystallography. The structure has been refined to 1.6 angstrom resolution with Rcryst and Rfree values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2, captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2, binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2, binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dirners or higher-order oligomeric states, and the CO2, bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.

  11. El Titulo IX y La Discriminacion por Sexo (Title IX and Sex Discrimination).

    ERIC Educational Resources Information Center

    Office for Civil Rights (ED), Washington, DC.

    Title IX of the Education Amendments of 1972 protects people from discrimination based on sex in education programs or activities that receive Federal financial assistance. This brochure outlines the responsibilities of education programs and activities covered by Title IX, the responsibilities of the Office for Civil Rights (OCR) in enforcing…

  12. 45 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... policy, ; 86.9 Distribution, 86.9(c) Notification of policy, ; 86.9(a) Publications, 86.9(b) Dress codes... 45 Public Welfare 1 2010-10-01 2010-10-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Public Welfare DEPARTMENT OF HEALTH...

  13. 34 CFR Subject Index to Title Ix... - Subject Index to Title IX Preamble and Regulation 1

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Distribution, 106.9(c) Notification of policy, ; 106.9(a) Publications, 106.9(b) Dress codes 106.31(b) (4) E... 34 Education 1 2010-07-01 2010-07-01 false Subject Index to Title IX Preamble and Regulation 1 Index Subject Index to Title IX Preamble and Regulation 1 Education Regulations of the Offices of...

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

    PubMed Central

    Dai, Hongyue; Mitchell, Melissa; Huntsman, Shane; Brantley, Stephen; Fenstermacher, David

    2016-01-01

    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. PMID:27057137

  15. Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

    ERIC Educational Resources Information Center

    Wang, Sheila C.; Zamble, Deborah B.

    2006-01-01

    A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

  16. Identification and characterization of a gene encoding a vertebrate-type carbonic anhydrase in cyanobacteria.

    PubMed Central

    Soltes-Rak, E; Mulligan, M E; Coleman, J R

    1997-01-01

    A gene (designated ecaA) encoding a vertebrate-like (alpha-type) carbonic anhydrase (CA) has been isolated from two disparate cyanobacteria, Anabaena sp. strain PCC 7120 and Synechococcus sp. strain PCC 7942. The deduced amino acid sequences correspond to proteins of 29 and 26 kDa, respectively, and revealed significant sequence similarity to human CAI and CAII, as well as Chlamydomonas CAHI, including conservation of most active-site residues identified in the animal enzymes. Structural similarities between the animal and cyanobacterial enzymes extend to the levels of antigenicity, as the Anabaena protein cross-reacts with antisera derived against chicken CAII. Expression of the cyanobacterial ecaA is regulated by CO2 concentration and is highest in cells grown at elevated levels of CO2. Immunogold localization using an antibody derived against the ecaA protein indicated an extracellular location. Preliminary analysis of Synechococcus mutants in which ecaA has been inactivated by insertion of a drug resistance cassette suggests that extracellular carbonic anhydrase plays a role in inorganic-carbon accumulation by maintaining equilibrium levels of CO2 and HCO3- in the periplasm. PMID:9006032

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

    PubMed

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

    2017-02-09

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

  18. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  19. Quantum supersymmetric Bianchi IX cosmology

    NASA Astrophysics Data System (ADS)

    Damour, Thibault; Spindel, Philippe

    2014-11-01

    We study the quantum dynamics of a supersymmetric squashed three-sphere by dimensionally reducing (to one timelike dimension) the action of D =4 simple supergravity for a S U (2 ) -homogeneous (Bianchi IX) cosmological model. The quantization of the homogeneous gravitino field leads to a 64-dimensional fermionic Hilbert space. After imposition of the diffeomorphism constraints, the wave function of the Universe becomes a 64-component spinor of spin(8,4) depending on the three squashing parameters, which satisfies Dirac-like, and Klein-Gordon-like, wave equations describing the propagation of a "quantum spinning particle" reflecting off spin-dependent potential walls. The algebra of the supersymmetry constraints and of the Hamiltonian one is found to close. One finds that the quantum Hamiltonian is built from operators that generate a 64-dimensional representation of the (infinite-dimensional) maximally compact subalgebra of the rank-3 hyperbolic Kac-Moody algebra A E3 . The (quartic-in-fermions) squared-mass term μ^ 2 entering the Klein-Gordon-like equation has several remarkable properties: (i) it commutes with all the other (Kac-Moody-related) building blocks of the Hamiltonian; (ii) it is a quadratic function of the fermion number NF; and (iii) it is negative in most of the Hilbert space. The latter property leads to a possible quantum avoidance of the singularity ("cosmological bounce"), and suggests imposing the boundary condition that the wave function of the Universe vanish when the volume of space tends to zero (a type of boundary condition which looks like a final-state condition when considering the big crunch inside a black hole). The space of solutions is a mixture of "discrete-spectrum states" (parametrized by a few constant parameters, and known in explicit form) and of continuous-spectrum states (parametrized by arbitrary functions entering some initial-value problem). The predominantly negative values of the squared-mass term lead to a "bottle

  20. Probing the surface of human carbonic anhydrase for clues towards the design of isoform specific inhibitors.

    PubMed

    Pinard, Melissa A; Mahon, Brian; McKenna, Robert

    2015-01-01

    The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3(-). Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of "selective drug targetability" and how these observations can be utilized to develop isoform selective CA inhibitors.

  1. Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

    PubMed Central

    Pinard, Melissa A.

    2015-01-01

    The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3−. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors. PMID:25811028

  2. In vivo evaluation of battery-operated light-emitting diode-based photodynamic therapy efficacy using tumor volume and biomarker expression as endpoints.

    PubMed

    Mallidi, Srivalleesha; Mai, Zhiming; Rizvi, Imran; Hempstead, Joshua; Arnason, Stephen; Celli, Jonathan; Hasan, Tayyaba

    2015-04-01

    In view of the increase in cancer-related mortality rates in low- to middle-income countries (LMIC), there is an urgent need to develop economical therapies that can be utilized at minimal infrastructure institutions. Photodynamic therapy (PDT), a photochemistry-based treatment modality, offers such a possibility provided that low-cost light sources and photosensitizers are available. In this proof-of-principle study, we focus on adapting the PDT light source to a low-resource setting and compare an inexpensive, portable, battery-powered light-emitting diode (LED) light source with a standard, high-cost laser source. The comparison studies were performed in vivo in a xenograft murine model of human squamous cell carcinoma subjected to 5-aminolevulinic acid-induced protoporphyrin IX PDT. We observed virtually identical control of the tumor burden by both the LED source and the standard laser source. Further insights into the biological response were evaluated by biomarker analysis of necrosis, microvessel density, and hypoxia [carbonic anhydrase IX (CAIX) expression] among groups of control, LED-PDT, and laser-PDT treated mice. There is no significant difference in the percent necrotic volume and CAIX expression in tumors that were treated with the two different light sources. These encouraging preliminary results merit further investigations in orthotopic animal models of cancers prevalent in LMICs.

  3. In vivo evaluation of battery-operated light-emitting diode-based photodynamic therapy efficacy using tumor volume and biomarker expression as endpoints

    PubMed Central

    Mallidi, Srivalleesha; Mai, Zhiming; Rizvi, Imran; Hempstead, Joshua; Arnason, Stephen; Celli, Jonathan; Hasan, Tayyaba

    2015-01-01

    Abstract. In view of the increase in cancer-related mortality rates in low- to middle-income countries (LMIC), there is an urgent need to develop economical therapies that can be utilized at minimal infrastructure institutions. Photodynamic therapy (PDT), a photochemistry-based treatment modality, offers such a possibility provided that low-cost light sources and photosensitizers are available. In this proof-of-principle study, we focus on adapting the PDT light source to a low-resource setting and compare an inexpensive, portable, battery-powered light-emitting diode (LED) light source with a standard, high-cost laser source. The comparison studies were performed in vivo in a xenograft murine model of human squamous cell carcinoma subjected to 5-aminolevulinic acid-induced protoporphyrin IX PDT. We observed virtually identical control of the tumor burden by both the LED source and the standard laser source. Further insights into the biological response were evaluated by biomarker analysis of necrosis, microvessel density, and hypoxia [carbonic anhydrase IX (CAIX) expression] among groups of control, LED-PDT, and laser-PDT treated mice. There is no significant difference in the percent necrotic volume and CAIX expression in tumors that were treated with the two different light sources. These encouraging preliminary results merit further investigations in orthotopic animal models of cancers prevalent in LMICs. PMID:25909707

  4. 77 FR 64401 - Order of Succession for HUD Region IX

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-19

    ... URBAN DEVELOPMENT Order of Succession for HUD Region IX AGENCY: Office of Field Policy and Management... Succession for the San Francisco Regional Office and its Field Offices (Region IX). This Order of Succession supersedes all previous Orders of Succession for HUD Region IX. DATES: Effective Date: October 9, 2012....

  5. The Restoration of Title IX: Implications for Higher Education.

    ERIC Educational Resources Information Center

    Sandler, Bernice R.

    This booklet helps institutions understand the restoration of Title IX of the Education Amendments of 1972 and changes resulting from the Civil Rights Restoration Act. Title IX prohibits sex discrimination in federally assisted education programs. A 1984 ruling held that Title IX covers only programs or activities funded with federal money. In…

  6. Title IX: With New Opportunities, Girls' Interest Rises

    ERIC Educational Resources Information Center

    Toporek, Bryan

    2012-01-01

    On June 23, 1972, President Richard M. Nixon signed into law Title IX of the Education Amendments of 1972, which prohibits gender discrimination in any federally financed education program or activity. Title IX is far-reaching, but the law is most often associated with school and college athletics. Title IX allows schools to prove their athletic…

  7. Open to All: Title IX at 30.

    ERIC Educational Resources Information Center

    Department of Education, Washington, DC. Office of the Secretary.

    This document is a report to the secretary of education on the findings of the Secretary's Commission on Opportunities in Athletics. The Commission was charged with examining Title IX. Starting in June 2002, the 15-member commission collected information, analyzed issues, and obtained broad public input directed at improving the application of…

  8. Title IX Enforcement Called 'Deeply Troubling'

    ERIC Educational Resources Information Center

    Lipka, Sara; Wolverton, Brad

    2007-01-01

    The government agency responsible for enforcing gender equity in college sports is falling down on the job, according to a report released by the National Women's Law Center. Over the past five years, the U.S. Education Department's Office for Civil Rights -- the administrative guardian of Title IX of the Education Amendments of 1972, the law that…

  9. Feminizing Science: The Alchemy of Title IX

    ERIC Educational Resources Information Center

    Hausman, Patricia

    2008-01-01

    The author scrutinizes the National Academy of Sciences report "Beyond Bias and Barriers: Fulfilling the Potential of Women in Academic Science and Engineering" and its dangerous call to place the sciences under the sledgehammer of Title IX. Her findings: A one-sided, inaccurate, and internally contradictory report prepared by a…

  10. Ares I-X Vibroacoustic Environments

    NASA Technical Reports Server (NTRS)

    Larsen, Curtis E.; Schuster, David M.; Kaufman, Daniel S.

    2009-01-01

    This paper provides a summary of the NASA Engineering and Safety Center (NESC) team recommendations and observations following participation with the Ares I-X Vibroacoustic (VA) Environments Panel in meetings at the Kennedy Space Center (KSC) and the Marshall Space Flight Center (MSFC) in March and April 2008, respectively.

  11. Mitochondrial proteomic profiling reveals increased carbonic anhydrase II in aging and neurodegeneration

    PubMed Central

    Pollard, Amelia; Shephard, Freya; Freed, James; Liddell, Susan; Chakrabarti, Lisa

    2016-01-01

    Carbonic anhydrase inhibitors are used to treat glaucoma and cancers. Carbonic anhydrases perform a crucial role in the conversion of carbon dioxide and water into bicarbonate and protons. However, there is little information about carbonic anhydrase isoforms during the process of ageing. Mitochondrial dysfunction is implicit in ageing brain and muscle. We have interrogated isolated mitochondrial fractions from young adult and middle aged mouse brain and skeletal muscle. We find an increase of tissue specific carbonic anhydrases in mitochondria from middle-aged brain and skeletal muscle. Mitochondrial carbonic anhydrase II was measured in the Purkinje cell degeneration (pcd5J) mouse model. In pcd5J we find mitochondrial carbonic anhydrase II is also elevated in brain from young adults undergoing a process of neurodegeneration. We show C.elegans exposed to carbonic anhydrase II have a dose related shorter lifespan suggesting that high CAII levels are in themselves life limiting. We show for the first time that the mitochondrial content of brain and skeletal tissue are exposed to significantly higher levels of active carbonic anhydrases as early as in middle-age. Carbonic anhydrases associated with mitochondria could be targeted to specifically modulate age related impairments and disease. PMID:27743511

  12. In-vitro study on ALA-induced endogenous protoporphyrin IX as photosensitizer for photodynamic tumor diagnosis and therapy

    NASA Astrophysics Data System (ADS)

    Ueberriegler, K.; Fiedler, D.; Verwanger, Thomas; Schnitzhofer, Gerlinde; Banieghbal, E.; Krammer, Barbara E.

    1998-07-01

    Photodynamic tumor diagnosis and therapy is efficiently carried out by endogenous protoporphyrin IX as photosensitizer, induced by external addition of the precursor 5-aminolevulinic acid (ALA). In the present study, PpIX localization and photodynamically induced damage was investigated in normal and transformed human fibroblasts. PpIX formation reaches its maximum after incubation for at least 20 h with 700 (mu) g/m1 ALA, and increases with the pH- value. ALA has to be given 20-30 times more than external PpIX in order to produce the same cytotoxic damage. As detected by Low Light Imaging, PpIX is generated in the mitochondria, released to the cytoplasm and distributed to cytoplasma and nuclear membranes.The nucleus is not stained. Intracellular targets of PpIX damage after irradiation are mainly mitochondria, ER and nuclear membrane. The organelles show a decomposition pattern, which resembles apoptotic morphology and occurs faster in the co-cultivated transformed than in the normal cells. ALA-treated hepatocytes produce micronuclei and chromosomal aberrations, which indicates some mutagenic potential. Expression studies of the (proto)oncogenes c-myc and bcl-2 sublethally treated fibroblasts by quantitative RT-PCR show high deviations from the constitutive expression level, which are accompanied by cell cycle disturbances, indicating a possible precursor role to apoptosis introduction.

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

    PubMed

    Ismail, Ibrahim Salihu

    2016-12-14

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

  14. Title IX Indian Education Toolkit. A Step-by-Step Guide to the Title IX Indian Education Formula Grant.

    ERIC Educational Resources Information Center

    Beller, Floyd

    Title IX is a federal Indian Education Formula Grant Program approved in 1972 and reauthorized five times, most recently in 1994. Title IX formula grants assist groups in providing sound educational programs and opportunities for American Indian and Alaska Native students. A 1997-98 survey of Title IX grantees revealed their need for help in…

  15. CO2 Removal using a Synthetic Analogue of Carbonic Anhydrase

    SciTech Connect

    Cordatos, Harry

    2010-09-14

    Project attempts to develop a synthetic analogue for carbonic anhydrase and incorporate it in a membrane for separation of CO2 from coal power plant flue gas. Conference poster presents result of first 9 months of project progress including concept, basic system architecture and membrane properties target, results of molecular modeling for analogue - CO2 interaction, and next steps of testing analogue resistance to flue gas contaminants.

  16. The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives

    PubMed Central

    Lionetto, Maria Giulia; Caricato, Roberto; Giordano, Maria Elena; Schettino, Trifone

    2016-01-01

    Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO2 to HCO3− and H+. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences. PMID:26797606

  17. Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

    PubMed Central

    Warden, Andrew C.; Williams, Michelle; Peat, Thomas S.; Seabrook, Shane A.; Newman, Janet; Dojchinov, Greg; Haritos, Victoria S.

    2015-01-01

    Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na+ hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated. PMID:26687908

  18. The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-terminal sequence extension.

    PubMed

    Martin-Eauclaire, Marie-France; Salvatierra, Juan; Bosmans, Frank; Bougis, Pierre E

    2016-09-01

    We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.

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

    PubMed Central

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

    2016-01-01

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

  20. Carbonic anhydrase isozymes IV and II in urinary membranes from carbonic anhydrase II-deficient patients.

    PubMed Central

    Sato, S; Zhu, X L; Sly, W S

    1990-01-01

    Carbonic anhydrase II (CA II) deficiency has been shown to be the primary defect in the recessively inherited syndrome of osteopetrosis with renal tubular acidosis. Until now, the absence of CA II in kidney of CA II-deficient patients has not been shown directly, and the status of the membrane-associated CA in kidney of CA II-deficient patients has been unclear. To address these questions, we analyzed urinary membranes and soluble fractions from normal and CA II-deficient subjects. The CA activity in membrane fractions of normal urine was found to comprise two components--(i) a vesicle-enclosed, sodium dodecyl sulfate (SDS)-sensitive fraction, which was shown immunochemically to be the 29-kDa CA II, and (ii) an SDS-resistant fraction, which was due to native and cleaved forms of the 35-kDa, membrane-anchored isozyme CA IV. Urinary membranes from CA II-deficient patients showed little or no SDS-sensitive activity and no immunoreactivity for CA II, providing direct evidence that their mutation, which produces CA II deficiency in erythrocytes, also affects CA II in kidney. CA IV activity and immunoreactivity were present in normal amounts in urinary membranes from CA II-deficient patients. We conclude from the enzymatic and immunological evidence presented that both CA II and CA IV are present in urinary membranes from normal subjects, that renal CA IV is present but renal CA II is absent in urinary membranes from patients with the CA II-deficiency syndrome, and that the methods presented should be useful in studying renal CA II and renal CA IV in other disorders of impaired bicarbonate reabsorption. Images PMID:2117271

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

    SciTech Connect

    Hu, P.Y.; Ernst, A.R.; Sly, W.S. ); Venta, P.J. ); Skaggs, L.A.; Tashian, R.E. )

    1994-04-01

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

  2. Dormant cancer cells accumulate high protoporphyrin IX levels and are sensitive to 5-aminolevulinic acid-based photodynamic therapy

    PubMed Central

    Nakayama, Taku; Otsuka, Shimpei; Kobayashi, Tatsuya; Okajima, Hodaka; Matsumoto, Kentaro; Hagiya, Yuichiro; Inoue, Keiji; Shuin, Taro; Nakajima, Motowo; Tanaka, Tohru; Ogura, Shun-ichiro

    2016-01-01

    Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in non-dormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor. PMID:27857072

  3. Early neoplastic and metastatic mammary tumours of transgenic mice detected by 5-aminolevulinic acid-stimulated protoporphyrin IX accumulation

    PubMed Central

    Dorward, A M; Fancher, K S; Duffy, T M; Beamer, W G; Walt, H

    2005-01-01

    A photodynamic technique for human breast cancer detection founded upon the ability of tumour cells to rapidly accumulate the fluorescent product protoporphyrin IX (PpIX) has been applied to transgenic mouse models of mammary tumorigenesis. A major goal of this investigation was to determine whether mouse mammary tumours are reliable models of human disease in terms of PpIX accumulation, for future mechanistic and therapeutic studies. The haeme substrate 5-aminolevulinic acid (5-ALA) (200 mg kg−1) was administered to mouse strains that develop mammary tumours of various histological subtypes upon expression of the transgenic oncogenes HRAS, Polyoma Virus middle T antigen, or Simian Virus 40 large T antigen in the mammary gland. Early neoplastic lesions, primary tumours and metastases showed consistent and rapid PpIX accumulation compared to the normal surrounding tissues, as evidenced by red fluorescence (635 nm) when the tumours were directly illuminated with blue light (380–440 nm). Detection of mouse mammary tumours at the stage of ductal carcinoma in situ by red fluorescence emissions suggests that enhanced PpIX synthesis is a good marker for early tumorigenic processes in the mammary gland. We propose the mouse models provide an ideal experimental system for further investigation of the early diagnostic and therapeutic potential of 5-ALA-stimulated PpIX accumulation in human breast cancer patients. PMID:16251872

  4. Removal of Zinc Form Carbonic Anhydrase: A Kinetics Experiment for Upper-Level Chemistry Laboratories

    ERIC Educational Resources Information Center

    Williams, Kathryn R.; Adhyaru, Bhavin

    2004-01-01

    An experiment on kinetics of deactivation of carbonic anhydrase by removal of zinc is demonstrated. Carbonic anhydrase, the enzyme that catalyzes the interconversion of carbon dioxide and bicarbonate, requires on Zn(II) ion in its active site, and removal of the zinc cofactor by complexion to another ligand leaves the apoenzyme, which is totally…

  5. Ares I-X 30 Day Report

    NASA Technical Reports Server (NTRS)

    Ess, Bob; Smith, Marshall

    2009-01-01

    This slide presentation represents the 30 day report on the Ares I-X test flight. Included in the review is information on the following areas: (1) Ground Systems, (2) Guidance, Navigation and Control, (3) Roll Response, (4) Vehicle Response, (5) Control System Performance, (6) Structural Damping, (7) Thrust Oscillation, (8) Stage Separation, (9) Connector Assessment, (10) USS Splashdown, (11) Data Recorder and (12) FS Hardware Assessment.

  6. On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase.

    PubMed

    Muther, T F

    1977-09-01

    Data are presented that support a nonenzymic mechanism for the staining obtained with the cobalt-bicarbonate method. The biochemically inactive apocarbonic anhydrase and Cu+2 apocarbonic anhydrase stain positively and this stain is inhibited by acetazolamide. The staining of the acetazolamide resistant carbonic anhydrase of male rat liver is inhibited by 10-6 M acetazolamide, at which concentration no biochemical inhibition is observed. There is no correlation between the biochemical and histochemical inhibitory potencies of a number of sulfonamides. The nonsulfonamide inhibitor, KCNO, does not inhibit staining. When incubations are performed in media exposed to atmospheres of increasing CO2 content, staining is not abolished until the atmospheric pCO2 approaches that generated by the medium itself. This finding renders the carbonic anhydrase catalyzed dehydration of HCO3- an improbable reaction for the staining. Studies with modified media show differences in staining patterns and in sensitivity to acetazolamide inhibition which question the specificity of the method for carbonic anhydrase.

  7. Antigen capsid-display on human adenovirus 35 via pIX fusion is a potent vaccine platform

    PubMed Central

    van der Helm, Esmeralda; Spek, Dirk; Vorthoren, Lars; Serroyen, Jan; Kuipers, Harmjan; Schuitemaker, Hanneke; Zahn, Roland; Custers, Jerome; Vellinga, Jort

    2017-01-01

    Durable protection against complex pathogens is likely to require immunity that comprises both humoral and cellular responses. While heterologous prime-boost regimens based on recombinant, replication-incompetent Adenoviral vectors (AdV) and adjuvanted protein have been able to induce high levels of concomitant humoral and cellular responses, complex manufacturing and handling in the field may limit their success. To combine the benefits of genetic and protein-based vaccination within one vaccine construct and to facilitate their use, we generated Human Adenovirus 35 (HAdV35) vectors genetically encoding a model antigen based on the Plasmodium falciparum (P. falciparum) circumsporozoite (CS) protein and displaying a truncated version of the same antigen (CSshort) via protein IX on the capsid, with or without a flexible glycine-linker and/or a 45Å-spacer. The four tested pIX-antigen display variants were efficiently incorporated and presented on the HAdV35 capsid irrespective of whether a transgene was encoded or not. Transgene-expression and producibility of the display-/expression vectors were not impeded by the pIX-display. In mice, the pIX-modified vectors induced strong humoral antigen-specific immunity that increased with the inclusion of the linker-/spacer molecules, exceeded the responses induced by the genetic, transgene-expressing HAdV35 vector, and surpassed recombinant protein in potency. In addition, the pIX- display/expression vectors elicited high antigen-specific cellular immune responses that matched those of the genetic HAdV35 vector expressing CS. pIX-modified display-/expression HAdV vectors may therefore be a valuable technology for the development of vaccines against complex pathogens, especially in resource-limited settings. PMID:28362809

  8. Activation of human factor IX (Christmas factor).

    PubMed Central

    Di Scipio, R G; Kurachi, K; Davie, E W

    1978-01-01

    Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its

  9. In Vivo Gene Therapy of Hemophilia B: Sustained Partial Correction in Factor IX-Deficient Dogs

    NASA Astrophysics Data System (ADS)

    Kay, Mark A.; Rothenberg, Steven; Landen, Charles N.; Bellinger, Dwight A.; Leland, Frances; Toman, Carol; Finegold, Milton; Thompson, Arthur R.; Read, M. S.; Brinkhous, Kenneth M.; Woo, Savio L. C.

    1993-10-01

    The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting. factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

  10. Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

    PubMed Central

    Liu, Yang-Sui; Li, Huan-Song; Qi, Dun-Feng; Zhang, Jun; Jiang, Xin-Chun; Shi, Kui; Zhang, Xiao-Jun; Zhang, Xin-Hui

    2014-01-01

    AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved. METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with 1 × 107 tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured. RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP. CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS. PMID:25024611

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

    PubMed

    Gilbert, A L; Guzmán, H M

    2001-09-01

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

  12. Evaluation of factor IX deficiency by interdigitated electrode (IDE)

    NASA Astrophysics Data System (ADS)

    Gopinath, Subash C. B.; Hashim, Uda; Uda, M. N. A.

    2017-03-01

    Factor IX deficiency is the main cause of hemophilia A and B. This a severe excessive bleeding disorder that can even kill the patient if not treated with the right prescription of Factor IX hormone to stop the bleeding. The bleeding can be caused by an injury or even a sudden bleeding in some very rare cases. To find the Factor IX effectiveness and to understand the deficiency more carefully for the future of medicine, experiments are conducted to test the Factor IX using the Interdigitated Electrode (IDE) and gold Nanoparticle with the help of Nanoelectrical technology.

  13. Antiproliferative and Apoptosis-inducing Effect of exo-Protoporphyrin IX based Sonodynamic Therapy on Human Oral Squamous Cell Carcinoma

    PubMed Central

    Lv, Yanhong; Zheng, Jinhua; Zhou, Qi; Jia, Limin; Wang, Chunying; Liu, Nian; Zhao, Hong; Ji, Hang; Li, Baoxin; Cao, Wenwu

    2017-01-01

    Sonodynamic therapy (SDT) is an innovative modality for cancer treatment. But the biological effect of SDT on oral squamous cell carcinoma has not been studied. Our previous study has shown that endo-Protoporphyrin IX based SDT (ALA-SDT) could induce apoptosis in human tongue squamous carcinoma SAS cells through mitochondrial pathway. Herein, we investigated the effect of exo- Protoporphyrin based SDT (PpIX-SDT) on SAS cells in vitro and in vivo. We demonstrated that PpIX-SDT increased the ratio of cells in the G2/M phase and induced 3–4 times more cell apoptosis compared to sonocation alone. PpIX-SDT caused cell membrane damage prior to mitochondria damage and upregulated the expression of Fas and Fas L, while the effect was suppressed if cells were pre-treated with p53 inhibitor. Additionally, we examined the SDT-induced cell apoptosis in two cell lines with different p53 status. The increases of p53 expression and apoptosis rate in wild-type p53 SAS cells were found in the SDT group, while p53-mutated HSC-3 cells did not show such increase. Our data suggest that PpIX-SDT suppress the proliferation of SAS cells via arresting cell cycle at G2/M phase and activating the extrinsic Fas-mediated membrane receptor pathway to induce apoptosis, which is regulated by p53. PMID:28102324

  14. IDH mutation status impact on in vivo hypoxia biomarkers expression: new insights from a clinical, nuclear imaging and immunohistochemical study in 33 glioma patients.

    PubMed

    Metellus, Philippe; Colin, Carole; Taieb, David; Guedj, Eric; Nanni-Metellus, Isabelle; de Paula, Andre Maues; Colavolpe, Cécile; Fuentes, Stephane; Dufour, Henry; Barrie, Marylin; Chinot, Olivier; Ouafik, L'houcine; Figarella-Branger, Dominique

    2011-12-01

    Mutations in the gene encoding isocitrate dehydrogenase enzyme isoforms 1 (IDH1) and 2 (IDH2) have recently been identified in a large proportion of glial tumors of the CNS, but their mechanistic role in tumor development remains unclear. Here, we assessed the actual impact of IDH1 and IDH2 mutations in patients harboring WHO grade II and III gliomas. We sequenced IDH1 at codon 132 and IDH2 at codon 172 in 33 patients with WHO grade II and III gliomas who benefited from a preoperative (18)F-FDG positron emission tomography (PET). Immunohistochemical expression of Hypoxia Inducible Factor-1alpha (HIF-1α), Carbonic Anhydrase IX (CAIX), Glucose Transporter 1 (GLUT1) and Caspase 3 active form (CASP3) along with the R132HIDH1 mutation was assessed in all cases as well as 1p/19q deletion status and p53 expression. HIF-1α expression was found in 15% of IDH-mutated compared to 7.7% of IDH-nonmutated tumors (P = 0.954). Also, GLUT-1 positive staining was found in 5% of IDH-mutated and in 7.1% of IDH-nonmutated tumors (P = 0.794). Finally, CA-IX expression was found in 15% of IDH-mutated and in 7.7% of IDH-nonmutated tumors (P = 0.484). The combined expression of these three hypoxic markers was found in two WHO grade III tumors, one of which was IDH-mutated whereas the other was IDH-nonmutated (P = 0.794). In IDH-mutated tumors, the median SUVmax ratio was 2.24 versus 2.15 in IDH-nonmutated tumors (P = 0.775). Together, these data question the actual relationship between IDH mutation status and in vivo hypoxic biomarkers expression in WHO grade II and III gliomas.

  15. Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro

    NASA Technical Reports Server (NTRS)

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

    1985-01-01

    The possible role of carbonic anhydrase in bone resorption induced by prostaglandin E2 (PGE2) was studied using an in vitro neonatal mouse calvarial culture system. PGE2 (10 to the -6th M) was effective in stimulating resorption, as assessed by calcium release into culture media. This enhanced resorption was accompanied by significant increases in calvarial carbonic anhydrase activity over control values at 48 and 96 h. At 48 h, bones treated with PGE2 had 20 percent more carbonic anhydrase activity than controls. By 96 h, treated bones contained 79 percent more carbonic anhydrase activity than controls. PGE2-induced bone resorption was inhibited by the carbonic anhydrase inhibitor acetazolamide in a dose-dependent fashion from 10 to the -5th to 10 to the -4th M with 77 percent inhibition observed at 10 to the -4th M. The acetazolamide analogue CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase, failed to inhibit PGE2-induced resorption. These results are consistent with the hypothesis that carbonic anhydrase is a necessary component of the osteoclastic bone resorptive mechanism.

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

    PubMed

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

    2017-12-01

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

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

    SciTech Connect

    Fisher, S. Z. Kovalevsky, A. Y.; Domsic, J.; Mustyakimov, M.; Silverman, D. N.; McKenna, R.; Langan, P.

    2010-11-01

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

  18. Histochemical carbonic anhydrase in rat inner medullary collecting duct

    NASA Technical Reports Server (NTRS)

    Kleinman, J. G.; Bain, J. L.; Fritsche, C.; Riley, D. A.

    1992-01-01

    Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.

  19. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice

    PubMed Central

    Verma, Dheeraj; Moghimi, Babak; LoDuca, Paul A.; Singh, Harminder D.; Hoffman, Brad E.; Herzog, Roland W.; Daniell, Henry

    2010-01-01

    To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin β-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20–25% of control animals survived after six to eight F.IX doses, 90–93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2–5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5–80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (∼40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment. PMID:20351275

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

    PubMed

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

    2009-02-10

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

  1. Carbonic anhydrase inhibition in glaucoma: hazard or benefit for the chronic lunger?

    PubMed

    Block, E R; Rostand, R A

    1978-01-01

    Carbonic anhydrase inhibitors, of proven value in the longterm management of glaucoma, have a number of troublesome side effects, most of which are well-known. However, their potential hazards to patients with chronic obstructive lung disease have received little attention. The author reviews the physiological effects of carbonic anhydrase inhibition on carbon dioxide metabolism and the implications for the chronic lung patient. The untoward pulmonary complications associated with the use of carbonic anhydrase inhibitors are of particular importance to ophthalmologists, since chronic lung disease and glaucoma, both common disorders in the elderly, frequently coexist in the same patient.

  2. Fish erythrocytes are bicarbonate permeable: problems with determining carbonic anhydrase activity using the modified boat technique.

    PubMed

    Heming, T A; Randall, D J

    1982-01-10

    Effects of foaming agents (blood plasma, bovine serum albumin, polyvinyl-pyrrolidinone) and defoaming agents (octanol, n-foam) on manometric determination of carbonic anhydrase activity in intact erythrocytes of fish were examined. Foaming agents abolished the activity of red cells. Defoaming agents increased the activity and negated any differences in carbonic anhydrase activity between whole blood (red cells in plasma) and "Cortland" blood (red cells in saline). It is concluded that effects of plasma on the carbonic anhydrase activity of intact erythrocytes of fish measured using the modified boat technique are largely nonspecific and related to reagent foaming during mixing, rather than to the action of a specific enzyme inhibitor.

  3. Participation of Two Carbonic Anhydrases of the Alpha Family in Photosynthetic Reactions in Arabidopsis thaliana.

    PubMed

    Zhurikova, E M; Ignatova, L K; Rudenko, N N; Mudrik, V A; Vetoshkina, D V; Ivanov, B N

    2016-10-01

    The expression of genes of two carbonic anhydrases (CA) belonging to the α-family, α-CA2 and α-CA4 (according to the nomenclature in N. Fabre et al. (2007) Plant Cell Environ., 30, 617-629), was studied in arabidopsis (Arabidopsis thaliana, var. Columbia) leaves. The expression of the At2g28210 gene coding α-CA2 decreased under increase in plant illumination, while the expression of the At4g20990 gene coding α-CA4 increased. Under conditions close to optimal for photosynthesis, in plants with gene At2g28210 knockout, the effective quantum yield of photosystem 2 and the light-induced accumulation of hydrogen peroxide in leaves were lower than in wild type plants, while the coefficient of non-photochemical quenching of leaf chlorophyll a fluorescence and the rate of CO2 assimilation in leaves were higher. In plants with At4g20990 gene knockout, the same characteristics changed in opposite ways relative to wild type. Possible mechanisms of the participation of α-CA2 and α-CA4 in photosynthetic reactions are discussed, taking into account that protons can be either consumed or released in the reactions they catalyze.

  4. Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

    PubMed Central

    Vilas, Gonzalo; Krishnan, Devishree; Loganathan, Sampath Kumar; Malhotra, Darpan; Liu, Lei; Beggs, Megan Rachele; Gena, Patrizia; Calamita, Giuseppe; Jung, Martin; Zimmermann, Richard; Tamma, Grazia; Casey, Joseph Roman; Alexander, Robert Todd

    2015-01-01

    Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins. PMID:25609088

  5. Carbonic anhydrase III: a new target for autoantibodies in autoimmune diseases.

    PubMed

    Robert-Pachot, Magali; Desbos, Agnes; Moreira, Annick; Becchi, Michel; Tebib, Jacques; Bonnin, Michel; Aitsiselmi, Tarik; Bienvenu, Jacques; Fabien, Nicole

    2007-07-01

    The objective of this study was to identify new autoantibodies that could be useful for the diagnosis of rheumatoid arthritis (RA) using immunoblotting on synovial membrane proteins which represent the best source of candidate RA autoantigens. A new target protein with a molecular weight of 26 kDa was found to be recognized by autoantibodies in RA sera and was identified using MALDI-TOF mass spectrometry and second-dimension electrophoresis as carbonic anhydrase III (CAIII). Three similar protein spots at 26 kDa were recognized by both human sera and monoclonal antibody (mAb) directed against CAIII on immunoblotting using the human recombinant CAIII. Interestingly, CAIII expression within the synovial membrane was not observed in non-RA patients and was differentially expressed among RA patients. The sensitivity of these new autoantibodies for RA, using an immunoenzymatic technique, was 17%. Specificity was high when comparing non-autoimmune diseases (100%), while it was found to be weak (67%) when comparing some other autoimmune diseases, and particularly systemic lupus erythematosus (SLE). In conclusion, this study demonstrates that these new autoantibodies against CAIII are not restricted to RA. However the expression of CAIII in the synovial membrane of RA warrants further investigation of the pathophysiological relevance of this finding.

  6. Enhancing protoporphyrin IX-induced PDT

    NASA Astrophysics Data System (ADS)

    Curnow, Alison; Pye, Andrew; Campbell, Sandra

    2009-06-01

    Photodynamic therapy (PDT) using porphyrin precursors is commonly used in dermatology. Evidence indicates that good clinical outcomes (associated with excellent cosmesis) can be achieved in superficial precancers and basal cell carcinoma (BCC), however, efficacy appears less favorable for thicker nodular BCC (nBCC) unless multiple PDT treatment cycles are performed. Enhancement is therefore required if nBCC lesions are to be treated effectively with a single PDT treatment. The most common technique currently being routinely employed clinically is the use of aminolevulinic acid (ALA) esters (usually methyl (MAL) or hexyl (HAL)). Standard dermatological PDT employing these porphyrin precursors already manipulates the normal heme biosynthesis pathway resulting in a temporary accumulation of the natural photosensitizer, protoporphyrin IX (PpIX). Further manipulation using iron chelating agents is possible however. In normal and malignant human cells in vitro, the novel iron chelating agent CP94 produced greater PPIX fluorescence when administered with ALA or MAL than either congener produced alone. CP94 was also significantly more effective than the clinically established iron chelating agent desferrioxamine (DFO). Topical application of ALA+CP94 to clinical nBCC lesions was a simple and safe treatment modification which produced a significant increase in clinical clearance when CP94 was included in the cream.

  7. Ares I-X Ascent Base Environments

    NASA Technical Reports Server (NTRS)

    Mobley, B. L.; Bender, R. L.; Canabal, F.; Smith, Sheldon D.

    2011-01-01

    Plume induced base heating environments were measured during the flight of the NASA Constellation Ares I-X developmental launch vehicle, successfully flown on October 28, 2009. The Ares IX first stage is a four segment Space Shuttle derived booster with base consisting of a flared aft skirt, deceleration and tumble motors, and a thermal curtain surrounding the first stage 7.2 area ratio nozzle. Developmental Flight Instrumentation (DFI) consisted of radiometers, calorimeters, pressure transducers and gas temperature probes installed on the aft skirt and nozzle to measure the base environments. In addition, thermocouples were also installed between the layers of the flexible thermal curtain to provide insight into the curtain response to the base environments and to assist in understanding curtain failure during reentry. Plume radiation environment predictions were generated by the Reverse Monte Carlo (RMC) code and the convective base heating predictions utilized heritage MSFC empirical methods. These predictions were compared to the DFI data and results from the flight videography. Radiation predictions agreed with the flight measured data early in flight but gauge failures prevented high altitude comparisons. The convective environment comparisons demonstrated the need to improve the prediction methodology; particularly for low altitude, local plume recirculation. The convective comparisons showed relatively good agreement at altitudes greater than 50,000 feet.

  8. Electron Impact Exciation of Fe IX

    NASA Astrophysics Data System (ADS)

    Tayal, Swaraj; Zatsarinny, Oleg

    2015-05-01

    Transition probabilities and electron impact excitation collision strengths and rates for astrophysically important extreme ultraviolet lines of Fe IX are calculated. The 322 fine-structure levels of the 3s2 3p6 , 3s2 3p5 3 d , 3 s 3p6 3 d , 3s2 3p5 4 s , and 3s2 3p4 3d2 configurations are included in our calculations. The collision strengths have been calculated using the B-spline Breit-Pauli R-matrix method for all fine-structure transitions among the 322 levels. The mass, Darwin, and spin-orbit relativistic effects are included in the Breit-Pauli Hamiltonian in the scattering calculation. The one-body and two-body relativistic operators are included in the multi-configuration Hartree-Fock calculations of transition probabilities. Several sets of non-orthogonal spectroscopic and correlation radial orbitals are used to obtain accurate description of Fe IX levels and to represent the scattering functions. The calculated excitation energies are in very good agreement with experiment and represents an improvement over the previous calculations. The present collision strengths show reasonable agreement with the previously available R-matrix and distorted-wave calculations. This research is supported by NASA grant from the Solar and Heliophysics Program.

  9. Ares I-X Ground Diagnostic Prototype

    NASA Technical Reports Server (NTRS)

    Schwabacher, Mark A.; Martin, Rodney Alexander; Waterman, Robert D.; Oostdyk, Rebecca Lynn; Ossenfort, John P.; Matthews, Bryan

    2010-01-01

    The automation of pre-launch diagnostics for launch vehicles offers three potential benefits: improving safety, reducing cost, and reducing launch delays. The Ares I-X Ground Diagnostic Prototype demonstrated anomaly detection, fault detection, fault isolation, and diagnostics for the Ares I-X first-stage Thrust Vector Control and for the associated ground hydraulics while the vehicle was in the Vehicle Assembly Building at Kennedy Space Center (KSC) and while it was on the launch pad. The prototype combines three existing tools. The first tool, TEAMS (Testability Engineering and Maintenance System), is a model-based tool from Qualtech Systems Inc. for fault isolation and diagnostics. The second tool, SHINE (Spacecraft Health Inference Engine), is a rule-based expert system that was developed at the NASA Jet Propulsion Laboratory. We developed SHINE rules for fault detection and mode identification, and used the outputs of SHINE as inputs to TEAMS. The third tool, IMS (Inductive Monitoring System), is an anomaly detection tool that was developed at NASA Ames Research Center. The three tools were integrated and deployed to KSC, where they were interfaced with live data. This paper describes how the prototype performed during the period of time before the launch, including accuracy and computer resource usage. The paper concludes with some of the lessons that we learned from the experience of developing and deploying the prototype.

  10. Cleavage and activation of human factor IX by serine proteases

    SciTech Connect

    Enfield, D.L.; Thompson, A.R.

    1984-10-01

    Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated /sup 125/I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa-tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of /sup 125/I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

  11. Tilting the Playing Field: Schools, Sports, Sex and Title IX.

    ERIC Educational Resources Information Center

    Gavora, Jessica

    This book suggests that Title IX of the Education Amendments is not creating more female athletes but instead eliminating some of the most prestigious men's sports programs in the name of gender equity. It shows how Title IX has affected every aspect of education, from kindergarten through graduate school, making profound changes in areas as…

  12. Beyond Title IX: Gender Equity Issues in Schools.

    ERIC Educational Resources Information Center

    NETWORK, Inc., Andover, MA.

    This document is one of a two-part set of publications. Both deal with equal education and provide a concise overview of Title IX and gender equity issues in education and steps to take to ensure nondiscrimination and equal education opportunity for all. Title IX of the Education Amendments of 1972 mandates that schools not deny any student…

  13. School Environment and Academic Achievement of Standard IX Students

    ERIC Educational Resources Information Center

    Lawrence, A. S. Arul; Vimala, A.

    2012-01-01

    The present study School Environment and Academic Achievement of standard IX students was probed to find the relationship between School Environment and Academic Achievement of standard IX students. Data for the study were collected using self-made School Environment Scale (SES). The investigator used stratified random sampling technique for…

  14. A License for Bias: Sex Discrimination, Schools, and Title IX.

    ERIC Educational Resources Information Center

    Morse, Susan Ed.

    This report discusses non-sports-related Title IX complaints filed with the Department of Education's Office for Civil Rights (OCR) from 1993-1997. Its purpose is to dispel the popular belief that Title IX is a sports-equity law and to determine the effectiveness of the legislation. The document examines the kinds of complaints filed, the status…

  15. The History, Uses, and Abuses of Title IX. 2016 Bulletin

    ERIC Educational Resources Information Center

    American Association of University Professors, 2016

    2016-01-01

    This report, an evaluation of the history and current uses of Title IX, is the result of a joint effort by a subcommittee that included members of the AAUP's Committee A on Academic Freedom and Tenure and the Committee on Women in the Academic Profession. The report identifies tensions between current interpretations of Title IX and the academic…

  16. A Model Community College Grievance Procedure for Title IX.

    ERIC Educational Resources Information Center

    Noonan, Roberta L.

    Through a review of the literature, analysis of eleven Title IX grievance plans, and interviews with four compliance officers, twelve criteria essential to an effective grievance procedure for use by students were identified and incorporated into a model Title IX grievance procedure for Moraine Valley Community College (Illinois). The twelve…

  17. ARES I-X USS Fracture Analysis Loads Spectra Development

    NASA Technical Reports Server (NTRS)

    Larsen, Curtis; Mackey, Alden

    2008-01-01

    This report describes the development of a set of bounding load spectra for the ARES I-X launch vehicle. These load spectra are used in the determination of the critical initial flaw size (CIFS) of the welds in the ARES I-X upper stage simulator (USS).

  18. Ares I-X Ground Diagnostic Prototype

    NASA Technical Reports Server (NTRS)

    Schwabacher, Mark; Martin, Rodney; Waterman, Robert; Oostdyk, Rebecca; Ossenfort, John; Matthews, Bryan

    2010-01-01

    Automating prelaunch diagnostics for launch vehicles offers three potential benefits. First, it potentially improves safety by detecting faults that might otherwise have been missed so that they can be corrected before launch. Second, it potentially reduces launch delays by more quickly diagnosing the cause of anomalies that occur during prelaunch processing. Reducing launch delays will be critical to the success of NASA's planned future missions that require in-orbit rendezvous. Third, it potentially reduces costs by reducing both launch delays and the number of people needed to monitor the prelaunch process. NASA is currently developing the Ares I launch vehicle to bring the Orion capsule and its crew of four astronauts to low-earth orbit on their way to the moon. Ares I-X will be the first unmanned test flight of Ares I. It is scheduled to launch on October 27, 2009. The Ares I-X Ground Diagnostic Prototype is a prototype ground diagnostic system that will provide anomaly detection, fault detection, fault isolation, and diagnostics for the Ares I-X first-stage thrust vector control (TVC) and for the associated ground hydraulics while it is in the Vehicle Assembly Building (VAB) at John F. Kennedy Space Center (KSC) and on the launch pad. It will serve as a prototype for a future operational ground diagnostic system for Ares I. The prototype combines three existing diagnostic tools. The first tool, TEAMS (Testability Engineering and Maintenance System), is a model-based tool that is commercially produced by Qualtech Systems, Inc. It uses a qualitative model of failure propagation to perform fault isolation and diagnostics. We adapted an existing TEAMS model of the TVC to use for diagnostics and developed a TEAMS model of the ground hydraulics. The second tool, Spacecraft Health Inference Engine (SHINE), is a rule-based expert system developed at the NASA Jet Propulsion Laboratory. We developed SHINE rules for fault detection and mode identification. The prototype

  19. Labeled factor IX kinetics in patients with hemophilia-B

    SciTech Connect

    Smith, K.J.; Thompson, A.R.

    1981-09-01

    Labeled factor IX was infused five time into four patients with hemophilia-B. Ten-minute plasma recovery average 35% (SD +/- 2) and the mean T 1/2 beta-phase elimination was 23 hr (+/- 5). No alteration in the postinfusion 125I-factor-IX could be detected by radioautography of plasma samples run on polyacrylamide gels or on crossed-immunoelectrophoresis. Label was excreted into the urine as free 125I-iodide. Kinetics were similar when the labeled preparation was infused alone or with a commercial concentrate containing unlabeled factor IX. Infusion of factor IX in man is best described by a two-compartment open pharmacokinetic model where factor IX is distributed in a space larger than the plasma volume.

  20. Inhibition of carbonic anhydrase II by steroidal and non-steroidal sulphamates.

    PubMed

    Ho, Y T; Purohit, A; Vicker, N; Newman, S P; Robinson, J J; Leese, M P; Ganeshapillai, D; Woo, L W L; Potter, B V L; Reed, M J

    2003-06-13

    Carbonic anhydrases (CAs) are expressed by many solid tumours where they may act to confer a growth advantage on malignant tissues. In this study we have examined the ability of a series of steroidal and non-steroidal sulphamates (originally developed as steroid sulphatase inhibitors) and related compounds to inhibit human CAII (hCAII) activity in vitro. Using a 96-well plate assay, oestrone-3-O-sulphamate (EMATE) and two coumarin-based sulphamate drugs (667 COUMATE and STX 118) were found to have IC(50) values of 25-59 nM for the inhibition of hCAII activity. These compounds therefore have a similar CAII inhibitory potency to that of acetazolamide (IC(50)=25 nM), a known hCAII inhibitor. Docking studies have been performed with selected compounds to the crystal structure of hCAII and excellent correlation of scores with biological activity was observed. This agrees with our recent observations when we were the first to report the inhibition of hCAII by STS inhibitors. These studies and initial results with docking to the crystal structure of the extracellular domain of hCAXII indicate that the STS sulphamate ester inhibitors should also be interesting candidates to pursue as inhibitors of CA isozymes that are over-expressed in human tumours.

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

    PubMed

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

    2013-06-22

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

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

    PubMed Central

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

    2013-01-01

    Osedax are gutless siboglinid worms that thrive on vertebrate bones lying on the ocean floor, mainly those of whales. The posterior body of female Osedax penetrates into the bone forming extensions known as ‘roots’, which host heterotrophic symbiotic bacteria in bacteriocytes beneath the epidermis. The Osedax root epithelium presumably absorbs bone collagen and/or lipids, which are metabolized by the symbiotic bacteria that in turn serve for Osedax's nutrition. Here, we show that Osedax roots express extremely high amounts of vacuolar-H+-ATPase (VHA), which is located in the apical membrane and in cytoplasmic vesicles of root and ovisac epithelial cells. The enzyme carbonic anhydrase (CA), which catalyses the hydration of CO2 into H+ and HCO3−, is also expressed in roots and throughout Osedax body. These results suggest Osedax roots have massive acid-secreting capacity via VHA, fuelled by H+ derived from the CA-catalysed hydration of CO2 produced by aerobic metabolism. We propose the secreted acid dissolves the bone carbonate matrix to then allow the absorption of bone-derived nutrients across the skin. In an exciting example of convergent evolution, this model for acid secretion is remarkably similar to mammalian osteoclast cells. However, while osteoclasts dissolve bone for repairing and remodelling, the Osedax root epithelium secretes acid to dissolve foreign bone to access nutrients. PMID:23760644

  3. Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells

    PubMed Central

    Jacobs, Andrea; Frei, Andreas P.; Ghigo, Dario; Wollscheid, Bernd; Riganti, Chiara

    2015-01-01

    Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer. We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells. We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment. PMID:25686827

  4. Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX.

    PubMed

    Wakamatsu, Jun-ichi; Hayashi, Nobutaka; Nishimura, Takanori; Hattori, Akihito

    2010-01-01

    The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation-reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.

  5. Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX.

    PubMed

    Wakamatsu, Jun-Ichi; Okui, Jun; Hayashi, Nobutaka; Nishimura, Takanori; Hattori, Akihito

    2007-12-01

    We examined the effects of exogenous myoglobin, a bivalent chelator, and nitrite on Zn protoporphyrin IX (ZPP) formation by using model systems. ZPP was formed in a model solution without addition of exogenous myoglobin. After incubation, the amount of ZPP in a model solution was increased but that of heme was not decreased compared with the amounts before incubation. Protoporphyrin IX (PPIX) instead of ZPP also accumulated in a model solution with addition of EDTA, but the amount of heme was not reduced. These results suggested that ZPP was not formed by the Fe-Zn substitution in heme but was formed by the insertion of Zn into PPIX, which was formed independently. The fact that the effects of various factors in model systems with/without addition of a bivalent chelator were similar suggested that ZPP formation was strongly affected by PPIX formation. Inhibition of PPIX formation by nitrite might be the reason for the low levels of ZPP in cured meats.

  6. Quantitative determination of Zn protoporphyrin IX, heme and protoporphyrin IX in Parma ham by HPLC.

    PubMed

    Wakamatsu, Jun-Ichi; Odagiri, Hiroko; Nishimura, Takanori; Hattori, Akihito

    2009-05-01

    We measured the contents of Zn protoporphyrin IX (ZPP), heme and protoporphyrin IX (PPIX) in Parma ham by simultaneous analysis using high-performance liquid chromatography (HPLC). Extraction with ethyl acetate-acetic acid (4:1) was suitable for the quantitative analysis of ZPP. The contents of heme, ZPP and PPIX in Parma ham were 15.0-29.9, 27.7-47.0 and 0.4-1.1μg/g, respectively, and total content of porphyrin was 43.7-76.6μg/g. The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60-70% of all porphyrins.

  7. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    PubMed

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.

  8. Evaluation of a carbonic anhydrase mimic for industrial carbon capture.

    PubMed

    Floyd, William C; Baker, Sarah E; Valdez, Carlos A; Stolaroff, Joshuah K; Bearinger, Jane P; Satcher, Joe H; Aines, Roger D

    2013-09-03

    Zinc(II) cyclen, a small molecule mimic of the enzyme carbonic anhydrase, was evaluated under rigorous conditions resembling those in an industrial carbon capture process: high pH (>12), nearly saturated salt concentrations (45% K2CO3) and elevated temperatures (100-130 °C). We found that the catalytic activity of zinc cyclen increased with increasing temperature and pH and was retained after exposure to a 45% w/w K2CO3 solution at 130 °C for 6 days. However, high bicarbonate concentrations markedly reduced the activity of the catalyst. Our results establish a benchmark level of stability and provide qualitative insights for the design of improved small-molecule carbon capture catalysts.

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

    PubMed

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

    1979-10-01

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

  10. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    PubMed

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  11. Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

    PubMed Central

    Poulin, Kathy L.; Lanthier, Robert M.; Smith, Adam C.; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L.; O'Meara, Ryan W.; Kothary, Rashmi; Lorimer, Ian A.; Parks, Robin J.

    2010-01-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions. PMID:20631131

  12. Carbonic anhydrase and control of breathing: different effects of benzolamide and methazolamide in the anaesthetized cat.

    PubMed Central

    Teppema, L; Berkenbosch, A; DeGoede, J; Olievier, C

    1995-01-01

    1. The effect of inhibition of erythrocyte carbonic anhydrase on the ventilatory response to CO2 was studied by administering benzolamide (70 mg kg-1, i.v.), an inhibitor which does not cross the blood-brain barrier, to carotid body denervated cats which were anaesthetized with chloralose-urethane. 2. In the same animals the effect on the ventilatory response to CO2 of subsequent inhibition of central nervous system (CNS) carbonic anhydrase was studied by infusing methazolamide (20 mg kg-1), an inhibitor which rapidly penetrates into brain tissue. 3. The results show that inhibition of erythrocyte carbonic anhydrase by benzolamide leads to a decrease in the slope of the normoxic CO2 response curve, and a decrease of the extrapolated arterial PCO2 at zero ventilation. 4. Inhibition of CNS carbonic anhydrase by methazolamide results in an increase in slope and alpha-intercept of the ventilatory CO2 response curve. 5. Using a mass balance equation for CO2 of a brain compartment, it is argued that inhibition of erythrocyte carbonic anhydrase results in a decrease in slope of the in vivo CO2 dissociation curve, which can explain the effects of benzolamide. 6. The changes in slope and intercept induced by methazolamide are discussed in relation to effects on neurones containing carbonic anhydrase, which may include central chemoreceptors. PMID:8576866

  13. Ares I-X USS Material Testing

    NASA Technical Reports Server (NTRS)

    Dawicke, David S.; Smith, Stephen W.; Raju, Ivatury S.

    2008-01-01

    An independent assessment was conducted to determine the critical initial flaw size (CIFS) for the flange-to-skin weld in the Ares I-X Upper Stage Simulator (USS). Material characterization tests were conducted to quantify the material behavior for use in the CIFS analyses. Fatigue crack growth rate, Charpy impact, and fracture tests were conducted on the parent and welded A516 Grade 70 steel. The crack growth rate tests confirmed that the material behaved in agreement with literature data and that a salt water environment would not significantly degrade the fatigue resistance. The Charpy impact tests confirmed that the fracture resistance of the material did not have a significant reduction for the expected operational temperatures of the vehicle.

  14. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    SciTech Connect

    Katzman, G.; Togasaki, R.K. ); Marcus, Y. ); Moroney, J.V. )

    1989-04-01

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

  15. Ares I-X Flight Test Philosophy

    NASA Technical Reports Server (NTRS)

    Davis, S. R.; Tuma, M. L.; Heitzman, K.

    2007-01-01

    In response to the Vision for Space Exploration, the National Aeronautics and Space Administration (NASA) has defined a new space exploration architecture to return humans to the Moon and prepare for human exploration of Mars. One of the first new developments will be the Ares I Crew Launch Vehicle (CLV), which will carry the Orion Crew Exploration Vehicle (CEV), into Low Earth Orbit (LEO) to support International Space Station (ISS) missions and, later, support lunar missions. As part of Ares I development, NASA will perform a series of Ares I flight tests. The tests will provide data that will inform the engineering and design process and verify the flight hardware and software. The data gained from the flight tests will be used to certify the new Ares/Orion vehicle for human space flight. The primary objectives of this first flight test (Ares I-X) are the following: Demonstrate control of a dynamically similar integrated Ares CLV/Orion CEV using Ares CLV ascent control algorithms; Perform an in-flight separation/staging event between an Ares I-similar First Stage and a representative Upper Stage; Demonstrate assembly and recovery of a new Ares CLV-like First Stage element at Kennedy Space Center (KSC); Demonstrate First Stage separation sequencing, and quantify First Stage atmospheric entry dynamics and parachute performance; and Characterize the magnitude of the integrated vehicle roll torque throughout the First Stage (powered) flight. This paper will provide an overview of the Ares I-X flight test process and details of the individual flight tests.

  16. ALA-induced PpIX fluorescence in epileptogenic tissue

    NASA Astrophysics Data System (ADS)

    Kleen, Jonathan K.; Valdes, Pablo A.; Harris, Brent T.; Holmes, Gregory L.; Paulsen, Keith D.; Roberts, David W.

    2011-03-01

    Astrogliotic tissue displays markedly increased levels of ALA-induced PpIX fluorescence, making it useful for fluorescence-guided resection in glioma surgery. In patients with temporal lobe epilepsy (TLE) and corresponding animal models, there are areas of astrogliosis that often co-localize with the epileptic focus, which can be resected to eliminate seizures in the majority of treated patients. If this epileptogenic tissue can exhibit PpIX fluorescence that is sufficiently localized, it could potentially help identify margins in epilepsy surgery. We tested the hypothesis that ALA-induced PpIX fluorescence could visually accentuate epileptogenic tissue, using an established animal model of chronic TLE. An acute dose of pilocarpine was used to induce chronic seizure activity in a rat. This rat and a normal control were given ALA, euthanized, and brains examined post-mortem for PpIX fluorescence and neuropathology. Preliminary evidence indicates increased PpIX fluorescence in areas associated with chronic epileptic changes and seizure generation in TLE, including the hippocampus and parahippocampal areas. In addition, strong PpIX fluorescence was clearly observed in layer II of the piriform cortex, a region known for epileptic reorganization and involvement in the generation of seizures in animal studies. We are further investigating whether ALA-induced PpIX fluorescence can consistently identify epileptogenic zones, which could warrant the extension of this technique to clinical studies for use as an adjuvant guidance technology in the resection of epileptic tissue.

  17. IBM PC/IX operating system evaluation plan

    NASA Technical Reports Server (NTRS)

    Dominick, Wayne D. (Editor); Granier, Martin; Hall, Philip P.; Triantafyllopoulos, Spiros

    1984-01-01

    An evaluation plan for the IBM PC/IX Operating System designed for IBM PC/XT computers is discussed. The evaluation plan covers the areas of performance measurement and evaluation, software facilities available, man-machine interface considerations, networking, and the suitability of PC/IX as a development environment within the University of Southwestern Louisiana NASA PC Research and Development project. In order to compare and evaluate the PC/IX system, comparisons with other available UNIX-based systems are also included.

  18. The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

    PubMed

    Swietach, Pawel; Patiar, Shalini; Supuran, Claudiu T; Harris, Adrian L; Vaughan-Jones, Richard D

    2009-07-24

    We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of approximately 300 microm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pH(i) (approximately 6.3) and reduced pH(e) (approximately 6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH(i) = approximately 6.6), whereas extracellular acidity was enhanced (pH(e) = approximately 6.6), so that radial pH(i) gradients were smaller and radial pH(e) gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH(e) indicates the enzyme is facilitating CO(2) excretion from cells (by converting vented CO(2) to extracellular H(+)), rather than facilitating membrane H(+) transport (such as H(+) associated with metabolically generated lactic acid). This latter process requires titration of exported H(+) ions with extracellular HCO(3)(-), which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH(e) will depend on the cellular CO(2)/lactic acid emission ratio (set by local oxygenation and membrane HCO(3)(-) uptake). Our results suggest that CO(2)-producing tumors may express CA9 to facilitate CO(2) excretion, thus raising pH(i) and reducing pH(e), which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

  19. Title IX and Intercollegiate Athletics in the Federal Appellate Courts: Myth vs. Reality.

    ERIC Educational Resources Information Center

    Pieronek, Catherine

    2000-01-01

    Focuses on the enforcement of the nondiscrimination provisions of Title IX in higher education, and shows how the courts have wrestled with the application of Title IX to college athletic programs over the past decade. Provides a history of Title IX enforcement and describes current approaches to evaluating Title IX compliance. (SLD)

  20. Preliminary X-ray crystallographic analysis of α-carbonic anhydrase from Thiomicrospira crunogena XCL-2.

    PubMed

    Díaz Torres, Natalia; González, Guillermo; Biswas, Shyamasri; Scott, Kathleen M; McKenna, Robert

    2012-09-01

    Thiomicrospira crunogena XCL-2 is a novel sulfur-oxidizing chemolithoautotroph that plays a significant role in the sustainability of deep-sea hydrothermal vent communities. This recently discovered gammaproteobacterium encodes and expresses four carbonic anhydrases (CAs) from three evolutionarily and structurally distinct CA families: an α-CA, two β-CAs and a γ-CA. In order to characterize and elucidate the physiological roles of these CAs, X-ray crystallographic structural studies have been initiated on the α-CA. The α-CA crystallized in space group C2. The crystals diffracted to a maximum resolution of 2.6 Å, with unit-cell parameters a = 127.1, b = 102.2, c = 105.0 Å, β = 127.3°, and a calculated Matthews coefficient of 2.04 Å(3) Da(-1) with four identical protein molecules in the crystallographic asymmetric unit. A preliminary solution was determined by molecular replacement with the PHENIX AutoMR wizard, which had an initial TFZ score of 17.9. Refinement of the structure is currently in progress.

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

    PubMed Central

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

    2017-01-01

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

  2. Mitochondrial gamma carbonic anhydrases are required for complex I assembly and plant reproductive development.

    PubMed

    Fromm, Steffanie; Braun, Hans-Peter; Peterhansel, Christoph

    2016-07-01

    Complex I of the mitochondrial electron transport chain (mETC) in plants contains an extra domain that is made up from proteins homologous to prokaryotic gamma-carbonic anhydrases (γCA). This domain has been suggested to participate in complex I assembly or to support transport of mitochondrial CO2 to the chloroplast. Here, we generated mutants lacking CA1 and CA2 - two out of three CA proteins in Arabidopsis thaliana. Double mutants were characterized at the developmental and physiological levels. Furthermore, the composition and activity of the mETC were determined, and mutated CA versions were used for complementation assays. Embryo development of double mutants was strongly delayed and seed development stopped before maturation. Mutant plants could only be rescued on sucrose media, showed severe stress symptoms and never produced viable seeds. By contrast, callus cultures were only slightly affected in growth. Complex I was undetectable in the double mutants, but complex II and complex IV were upregulated concomitant with increased oxygen consumption in mitochondrial respiration. Ectopic expression of inactive CA variants was sufficient to complement the mutant phenotype. Data indicate that CA proteins are structurally required for complex I assembly and that reproductive development is dependent on the presence of complex I.

  3. Molecular mechanism of kNBC1-carbonic anhydrase II interaction in proximal tubule cells.

    PubMed

    Pushkin, Alexander; Abuladze, Natalia; Gross, Eitan; Newman, Debra; Tatishchev, Sergei; Lee, Ivan; Fedotoff, Olga; Bondar, Galyna; Azimov, Rustam; Ngyuen, Matt; Kurtz, Ira

    2004-08-15

    We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L(958)DDV and D(986)NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. Carbonic anhydrase inhibition for the management of cerebral ischemia: in vivo evaluation of sulfonamide and coumarin inhibitors.

    PubMed

    Di Cesare Mannelli, Lorenzo; Micheli, Laura; Carta, Fabrizio; Cozzi, Andrea; Ghelardini, Carla; Supuran, Claudiu T

    2016-12-01

    Ischemia of brain areas is a global health problem, causing death or long-term disability. Current pharmacological options have limited impact on ischemic damages. Recently, a relationship between hypoxia and carbonic anhydrase (CA) over-expression has been highlighted suggesting CA inhibition as a possible target. This study aimed to evaluate the pharmacological profile of sulfonamide and coumarin CA inhibitors in rats underwent permanent middle cerebral artery occlusion (pMCAO). The neurological score of pMCAO rats was dramatically reduced 24 h after occlusion. Repeated subcutaneous injections of the CA inhibitors 4 and 7 (1 mg kg(-1)) were able to increase the neurological score by 40%. Compound 7 showed the tendency to reduce the volume of hemisphere infarction. The standard CA inhibitor acetazolamide was ineffective. The properties of novel CA inhibitors to improve neurological functionalities after cerebral ischemic insult are shown. The CA involvement in cerebral hypoxic phenomena deserves deeper investigations.

  8. Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

    PubMed

    Dekaminaviciute, Dovile; Kairys, Visvaldas; Zilnyte, Milda; Petrikaite, Vilma; Jogaite, Vaida; Matuliene, Jurgita; Gudleviciene, Zivile; Vullo, Daniela; Supuran, Claudiu T; Zvirbliene, Aurelija

    2014-12-01

    Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

  9. 29. Photocopy of 1921 photograph. Glass Negative Box IX, Tower ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    29. Photocopy of 1921 photograph. Glass Negative Box IX, Tower Grove, Missouri Botanical Garden. ITALIAN GARDEN AND NEW PALM HOUSE (DEMOLISHED), LOOKING NORTHEAST - Missouri Botanical Garden, 2345 Tower Grove Avenue, Saint Louis, Independent City, MO

  10. 26. Photocopy of August 1918 photograph. Glass Negative Box IX, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    26. Photocopy of August 1918 photograph. Glass Negative Box IX, Tower Grove, Missouri Botanical Garden. ITALIAN GARDEN, LOOKING SOUTHWEST - Missouri Botanical Garden, 2345 Tower Grove Avenue, Saint Louis, Independent City, MO

  11. Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice.

    PubMed

    Juzenas, Petras; Juzeniene, Asta; Stakland, Silje; Iani, Vladimir; Moan, Johan

    2002-09-27

    No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.

  12. Anisotropic Bianchi types VIII and IX locally rotationally symmetric cosmologies

    SciTech Connect

    Assad, M.J.D.; Soares, I.D.

    1983-10-15

    We present a class of exact cosmological solutions of Einstein-Maxwell equations, which are anisotropic and spatially homogeneous of Bianchi types VIII and IX, and class IIIb in the Stewart-Ellis classification of locally rotationally symmetric models. If we take the electromagnetic field equal to zero, a class of Bianchi types VIII/IX spatially homogeneous anisotropic cosmological solutions with perfect fluid is obtained.

  13. Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland.

    PubMed Central

    Fernley, R T; Coghlan, J P; Wright, R D

    1988-01-01

    Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus. Images Fig. 4. Fig. 6. Fig. 7. PMID:3124821

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

    PubMed

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

    2015-11-15

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

  15. Carbonic anhydrase enzymes regulate mast cell–mediated inflammation

    PubMed Central

    Soteropoulos, Patricia

    2016-01-01

    Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine–mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2–associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy–like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell–mediated inflammation. PMID:27526715

  16. Characterization of carbonic anhydrase-inhibitor noncovalent complexes

    SciTech Connect

    Cheng, Xueheng; Chen, R.; Bruce, J.E.

    1995-12-31

    Competitive binding of two mixtures of inhibitors to bovine carbonic anhydrase H (BCAII) was studied using electrospray ionization mass spectrometry (ESI-MS). The first mixture contained inhibitors with hydrocarbon/fluorohydrocarbon linker groups and with an 800 fold span of binding constants. The second contained inhibitors with dipeptide extensions synthesized using the solid phase method. Noncovalent enzyme-inhibitor complexes were observed from solutions in 10 mM ammonia acetate having abundances consistent with their relative binding constants measured in solution. The inhibitor with highest affinity was readily identified in an equimolar mixture. The inhibitors with very low affinity were identified to form specific complexes as well. Several control experiments including acidifying the solution or removing the Zn metal from the enzyme resulted in the disappearance of the enzyme-inhibitor complexes, (mass spectrometrically) observed complexation and characterization of biomolecular binding. Good correlation between gas phase inhibitor ion abundances and their binding constants in solution were observed. Structural information and relative binding constants of masses were obtained using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) through multi-stage dissociation experiments. These results support the role of ESI-FTICR-MS in the study of specific noncovalent associations from solution, and show that its unique capabilities can be exploited to extend studies to large mixtures of inhibitors in drug leads discovery.

  17. Quaternary ammonium sulfanilamide: a membrane-impermeant carbonic anhydrase inhibitor.

    PubMed

    Henry, R P

    1987-05-01

    A novel carbonic anhydrase (CA) inhibitor, quaternary ammonium sulfanilamide (QAS), was tested for potency as a CA inhibitor and for its ability to be excluded from permeating biological membranes. Inhibitor titration plots of QAS vs. pure bovine CA II and CA from the gills of the blue crab, Callinectes sapidus, yielded Ki values of approximately 15 microM; thus QAS is a relatively weak but effective CA inhibitor. Permeability of the QAS was directly tested by two independent methods. The inhibitor was excluded from human erythrocytes incubated in 5 mM QAS for 24 h as determined using an 18O-labeled mass spectrometer CA assay for intact cells. Also QAS injected into the hemolymph of C. sapidus (1 or 10 mM) did not cross the basal membrane of the gill. The compound was cleared from the hemolymph by 96 h after injection, and at no time during that period could the QAS be detected in homogenates of gill tissue. Total branchial CA activity was only slightly reduced following the QAS injection. These data indicate that QAS is a CA inhibitor to which biological membranes are impermeable and that can be used in vivo or in vitro in the study of membrane-associated CA.

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

    PubMed

    Arabaci, Betul; Gulcin, Ilhami; Alwasel, Saleh

    2014-07-10

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

  19. Dynamics of oligomer formation by denatured carbonic anhydrase II.

    PubMed

    Prokhorov, Dmitry A; Timchenko, Alexander A; Uversky, Vladimir N; Khristoforov, Vladimir S; Kihara, Hiroshi; Kimura, Kazumoto; Kutyshenko, Viktor P

    2008-05-01

    Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

  20. Carbonic anhydrase and acetylcholinesterase inhibitory effects of carbamates and sulfamoylcarbamates.

    PubMed

    Göçer, Hülya; Akincioğlu, Akın; Göksu, Süleyman; Gülçin, İlhami; Supuran, Claudiu T

    2015-04-01

    Carbonic anhydrases (CA), as a family of metalloenzymes, are found in almost every type of tissue and play an important role in catalyzing the equilibration of carbon dioxide and carbonic acid. In this study, a series of carbamate derivative was synthesized, and their inhibition effects on hCA I, hCA II and acetylcholinesterase (AChE) enzymes were investigated. They were determined to be very good inhibitor against for both isoenzymes (hCA I and hCA II) and AChE. The hCA I and hCA II were effectively inhibited by the carbamate derivatives, with inhibition constants (Ki) in the range of 194.4-893.5 nM (for hCA I) and 103.9-835.7 nM (for hCA II). On the other hand, Ki parameters of these compounds for AChE enzyme inhibition were determined in the range of 12.0-61.3 nM. The results clearly showed that both CA isoenzymes and AChE were inhibited by carbamate derivatives at the nM levels.

  1. Electropolymerized carbonic anhydrase immobilization for carbon dioxide capture.

    PubMed

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

    2014-06-17

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

  2. Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements1[OPEN

    PubMed Central

    Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; Ries, Amber; Böhmer, Maik; You, Lei; Xiao, Chuanlei; Engineer, Cawas B.; Boron, Walter F.; Schroeder, Julian I.

    2015-01-01

    Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3− concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3− concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified. PMID:26243620

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

    PubMed

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

    2016-06-01

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

  4. Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Schlossmacher, Ute; Grebenjuk, Vlad A; Ushijima, Hiroshi; Wang, Xiaohong

    2013-11-01

    Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.

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

    PubMed Central

    Asahi, Masumi G; Bobarnac Dogaru, Gabriela L; Onishi, Spencer M; Gallemore, Ron P

    2015-01-01

    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. PMID:26664246

  6. Carbonic anhydrase inhibitors: Synthesis, characterization and inhibition activities of furan sulfonylhydrazones against carbonic anhydrase I (hCA I)

    NASA Astrophysics Data System (ADS)

    Gündüzalp, Ayla Balaban; Parlakgümüş, Gökhan; Uzun, Demet; Özmen, Ümmuhan Özdemir; Özbek, Neslihan; Sarı, Musa; Tunç, Tuncay

    2016-02-01

    The methane sulfonic acide hydrazide (1) was used to obtain furan sulfonylhydrazones; 2-acetylfuranmethanesulfonylhydrazone (2), 2-furaldehydemethanesulfonylhydrazone (3), 5-nitro-2-furaldehydemethanesulfonylhydrazone (4). The structures of furan sulfonylhydrazones were determined by using elemental analysis, FT-IR, 1H NMR, 13C NMR and UV-vis methods. The structure of 5-nitro-2-furaldehydemethanesulfonylhydrazone (4) was also supported with X-ray difraction method and found that compound 4 was crystallized in triclinic, space group P 1 bar . In order to gain insight into the structure of the compounds, we performed computational studies by using 6-311G(d,p) basic set in which B3LYP correlation function was implemented. The geometry of the sulfonylhydrazones were optimized at DFT method with Gaussian 09 program package and the global reactivity descriptors were also calculated by this basic set. The enzyme inhibition activities of the sulfonylhydrazones were investigated on carbonic anhydrase I (hCA I) isoenzyme and their activity parameters (Km, IC50 and Ki) were calculated by spectrophotometric method. And also, their inhibitor effects were also investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Inhibition results show that compound 4 containing electron withdrawing group (NO2) has higher inhibition effect on hCA I isoenzyme than other's.

  7. Inhibition of carbonic anhydrase augments GABAA receptor-mediated analgesia via a spinal mechanism of action

    PubMed Central

    Asiedu, Marina N.; Mejia, Galo L.; Hübner, Christian A.; Kaila, Kai; Price, Theodore J.

    2014-01-01

    Peripheral nerve injury negatively influences spinal GABAergic networks via a reduction in the neuron-specific K+-Cl- cotransporter KCC2. This process has been linked to the emergence of neuropathic allodynia. In vivo pharmacological and modeling studies show that a loss of KCC2 function results in a decrease in the efficacy of GABAA -mediated spinal inhibition. One potential strategy to mitigate this effect entails inhibition of carbonic anhydrase activity to reduce HCO3- -dependent depolarization via GABAA receptors when KCC2 function is compromised. We have tested this hypothesis here. Our results show that, similarly to when KCC2 is pharmacologically blocked, peripheral nerve injury causes a loss of analgesic effect for neurosteroid GABAA allosteric modulators at maximally effective doses in naïve mice in the tail flick test. Remarkably, inhibition of carbonic anhydrase activity with intrathecal acetazolamide rapidly restores an analgesic effect for these compounds suggesting an important role of carbonic anhydrase activity in regulating GABAA -mediated analgesia after peripheral nerve injury. Moreover, spinal acetazolamide administration leads to a profound reduction in the mouse formalin pain test indicating that spinal carbonic anhydrase inhibition produces analgesia when primary afferent activity is driven by chemical mediators. Finally, we demonstrate that systemic administration of acetazolamide to rats with peripheral nerve injury produces an anti-allodynia effect by itself and an enhancement of the peak analgesic effect with a change in the shape of the dose response curve of the α1-sparing benzodiazepine L-838,417. Thus, carbonic anhydrase inhibition mitigates the negative effects of loss of KCC2 function after nerve injury in multiple species and through multiple administration routes resulting in an enhancement of analgesic effects for several GABAA allosteric modulators. We suggest that carbonic anhydrase inhibitors, many of which are clinically

  8. Long-Acting Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Children

    PubMed Central

    Chambost, Hervé; Male, Christoph; Lambert, Thierry; Halimeh, Susan; Chernova, Tatiana; Mancuso, Maria Elisa; Curtin, Julie; Voigt, Christine; Li, Yanyan; Jacobs, Iris; Santagostino, Elena

    2016-01-01

    Summary A global phase 3 study evaluated the pharmacokinetics, efficacy and safety of a recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 27 previously treated male children (1–11 years) with severe and moderately severe haemophilia B (factor IX [FIX] activity ≤2 IU/dl). All patients received routine prophylaxis once every seven days for up to 77 weeks, and treated any bleeding episodes on-demand. The mean terminal half-life of rIX-FP was 91.4 hours (h), 4.3-fold longer than previous FIX treatment and clearance was 1.11 ml/h/kg, 6.4-fold slower than previous FIX treatment. The median (Q1, Q3) annualised spontaneous bleeding rate was 0.00 (0.00, 0.91) and was similar between the <6 years and ≥6 years age groups, with a weekly median prophylactic dose of 46 IU/kg. In addition, patients maintained a median trough level of 13.4 IU/dl FIX activity on weekly prophylaxis. Overall, 97.2% of bleeding episodes were successfully treated with one or two injections of rIX-FP (95% CI: 92% to 99%), 88.7% with one injection, and 96% of the treatments were rated effective (excellent or good) by the Investigator. No patient developed FIX inhibitors and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in children with haemophilia B with weekly prophylaxis. Routine prophylaxis with rIX-FP at treatment intervals of up to 14 days are currently being investigated in children with severe and moderately severe haemophilia B. Clinicaltrials.gov (NCT01662531) PMID:27583313

  9. Carbonic anhydrase binding site parameterization in OPLS-AA force field.

    PubMed

    Bernadat, Guillaume; Supuran, Claudiu T; Iorga, Bogdan I

    2013-03-15

    The parameterization of carbonic anhydrase binding site in OPLS-AA force field was performed using quantum chemistry calculations. Both OH2 and OH(-) forms of the binding site were considered, showing important differences in terms of atomic partial charges. Three different parameterization protocols were used, and the results obtained highlighted the importance of including an extended binding site in the charge calculation. The force field parameters were subsequently validated using standard molecular dynamics simulations. The results presented in this work should greatly facilitate the use of molecular dynamics simulations for studying the carbonic anhydrase, and more generally, the metallo-enzymes.

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

    PubMed

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

    2016-01-07

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

  11. Ares I-X Flight Test - The Future Begins Here

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.

    2008-01-01

    In less than two years, the National Aeronautics and Space Administration (NASA) will launch the Ares I-X mission. This will be the first flight of the Ares I crew launch vehicle, which, together with the Ares V cargo launch vehicle, will eventually send humans to the Moon, Mars, and beyond. As the countdown to this first Ares mission continues, personnel from across the Ares I-X Mission Management Office (MMO) are finalizing designs and fabricating vehicle hardware for an April 2009 launch. This paper will discuss the hardware and programmatic progress of the Ares I-X mission. Like the Apollo program, the Ares launch vehicles will rely upon extensive ground, flight, and orbital testing before sending the Orion crew exploration vehicle into space with humans on board. The first flight of Ares I, designated Ares I-X, will be a suborbital development flight test. Ares I-X gives NASA its first opportunity to gather critical data about the flight dynamics of the integrated launch vehicle stack; understand how to control its roll during flight; better characterize the severe stage separation environments that the upper stage engine will experience during future operational flights; and demonstrate the first stage recovery system. NASA also will begin modifying the launch infrastructure and fine-tuning ground and mission operations, as the agency makes the transition from the Space Shuttle to the Ares/Orion system.

  12. Targeting of the epidermal growth factor receptor with mesoporphyrin IX-peptide conjugates.

    PubMed

    Fontenot, Krystal R; Ongarora, Benson G; LeBlanc, Logan E; Zhou, Zehua; Jois, Seetharama D; Vicente, M Graça H

    2016-01-01

    The synthesis and in vitro evaluation of four mesoporphyrin IX-peptide conjugates designed to target EGFR, over-expressed in colorectal and other cancers, are reported. Two peptides with known affinity for EGFR, LARLLT (1) and GYHWYGYTPQNVI (2), were conjugated to mesoporphyrin IX (MPIX, 3) via one or both the propionic side chains, directly (4, 5) or with a triethylene glycol spacer (7, 8). The conjugates were characterized using NMR, MS, CD, SPR, UV-vis and fluorescence spectroscopies. Energy minimization and molecular dynamics suggest different conformations for the conjugates. SPR studies show that conjugate 4, bearing two LARLLT with no PEG spacers, has the greatest affinity for binding to EGFR, followed by conjugate 7 with two PEG and two LARLLT sequences. Molecular modeling and docking studies suggest that both conjugates 4 and 7 can bind to monomer and dimer EGFR in open and closed conformations. The cytotoxicity and cellular targeting ability of the conjugates were investigated in human HEp2 cells over-expressing EGFR. All conjugates showed low dark- and photo-toxicities. The cellular uptake was highest for conjugates 4 and 8 and lowest for 7 bearing two LARLLT linked via PEG groups, likely due to decreased hydrophobicity. Among the conjugates investigated 4 is the most efficient EGFR-targeting agent, and therefore the most promising for the detection of cancers that over-express EGFR.

  13. Targeting of the epidermal growth factor receptor with mesoporphyrin IX-peptide conjugates

    PubMed Central

    Fontenot, Krystal R.; Ongarora, Benson G.; LeBlanc, Logan E.; Zhou, Zehua; Jois, Seetharama D.; Vicente, M. Graça H.

    2016-01-01

    The synthesis and in vitro evaluation of four mesoporphyrin IX-peptide conjugates designed to target EGFR, over-expressed in colorectal and other cancers, are reported. Two peptides with known affinity for EGFR, LARLLT (1) and GYHWYGYTPQNVI (2), were conjugated to mesoporphyrin IX (MPIX, 3) via one or both the propionic side chains, directly (4, 5) or with a triethylene glycol spacer (7, 8). The conjugates were characterized using NMR, MS, CD, SPR, UV-vis and fluorescence spectroscopies. Energy minimization and molecular dynamics suggest different conformations for the conjugates. SPR studies show that conjugate 4, bearing two LARLLT with no PEG spacers, has the greatest affinity for binding to EGFR, followed by conjugate 7 with two PEG and two LARLLT sequences. Molecular modeling and docking studies suggest that both conjugates 4 and 7 can bind to monomer and dimer EGFR in open and closed conformations. The cytotoxicity and cellular targeting ability of the conjugates were investigated in human HEp2 cells over-expressing EGFR. All conjugates showed low dark- and photo-toxicities. The cellular uptake was highest for conjugates 4 and 8 and lowest for 7 bearing two LARLLT linked via PEG groups, likely due to decreased hydrophobicity. Among the conjugates investigated 4 is the most efficient EGFR-targeting agent, and therefore the most promising for the detection of cancers that over-express EGFR. PMID:27738394

  14. Legionella pneumophila Carbonic Anhydrases: Underexplored Antibacterial Drug Targets

    PubMed Central

    Supuran, Claudiu T.

    2016-01-01

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

  15. Carbonic anhydrase and chemoreception in the cat carotid body.

    PubMed

    Iturriaga, R; Lahiri, S; Mokashi, A

    1991-10-01

    To test the hypothesis that CO2 and O2 chemoreception in the carotid body (CB) may depend on its carbonic anhydrase (CA) activity, we used an in vitro cat CB preparation and studied the effects of methazolamide, a permeable CA inhibitor (pK 7.3), on the chemosensory responses to CO2, O2, and nicotine. The isolated CB was perfused and superfused with Tyrode solution, free of CO2-HCO3-, at 36.0 +/- 0.5 degrees C. The frequency of chemosensory discharges was recorded from the whole carotid sinus nerve. The responses to bolus injections (0.3-0.5 ml) of Tyrode solution equilibrated with PCO2 of 38-110 Torr, switching from HEPES to CO2-HCO3- Tyrode (PCO2 = 25-60 Torr) for about 3 min, hypoxic Tyrode (PO2 = 25-30 Torr) for 2-8 min, perfusate flow interruptions for approximately 4 min, and bolus injections of nicotine (4 nmol) were studied before, during, and after perfusion (30-45 min) with methazolamide (42.4 microM). Methazolamide reversibly inhibited, delayed, and reduced the responses to transient CO2 stimulus, diminished the onset of but not the late response to prolonged CO2 stimulus, and delayed but did not decrease the responses to hypoxia and perfusate interruption. The response to nicotine did not change. The results indicated that CA in the glomus cells played a crucial role primarily in the speed and magnitude of the initial response to CO2 stimulus and indirectly influenced O2 chemoreception. These effects were upstream from the nicotine receptor-mediated sensory response.

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Improved murine glioma detection following modified diet and photobleaching of skin PpIX fluorescence

    NASA Astrophysics Data System (ADS)

    Gibbs, Summer L.; O'Hara, Julia A.; Hoopes, P. Jack; Pogue, Brian W.

    2007-02-01

    The Aminolevulinic Acid (ALA) - Protoporphyrin IX (PpIX) system is unique in the world of photosensitizers in that the prodrug ALA is enzymatically transformed via the tissue of interest into fluorescently detectable levels of PpIX. This system can be used to monitor cellular metabolism of tumor tissue for applications such as therapy monitoring. Detecting PpIX fluorescence noninvasively has proven difficult due to the high levels of PpIX produced in the skin compared to other tissue both with and without ALA administration. In the current study, methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration have been examined. Use of a purified diet is found to decrease both skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration, while addition of a broad spectrum antibiotic to the water shows little effect. Following ALA administration, improved brain tumor detection is seen when skin PpIX fluorescence is photobleached via blue light prior to transmission spectroscopic measurements of tumor bearing and control animals. Both of these methods to decrease skin PpIX autofluorescence and skin PpIX fluorescence following ALA administration are shown to have a large effect on the ability to detect tumor tissue PpIX fluorescence noninvasively in vivo.

  19. Ares I-X: First Flight of a New Era

    NASA Technical Reports Server (NTRS)

    Davis, Stephen R.; Askins, Bruce R.

    2010-01-01

    Since 2005, NASA s Constellation Program has been designing, building, and testing the next generation of launch and space vehicles to carry humans beyond low-Earth orbit (LEO). The Ares Projects at Marshall Space Flight Center (MSFC) are developing the Ares I crew launch vehicle and Ares V cargo launch vehicle. On October 28, 2009, the first development flight test of the Ares I crew launch vehicle, Ares I-X, lifted off from a launch pad at Kennedy Space Center (KSC) on successful suborbital flight. Basing exploration launch vehicle designs on Ares I-X information puts NASA one step closer to full-up "test as you fly," a best practice in vehicle design. Although the final Constellation Program architecture is under review, the Ares I-X data and experience in vehicle design and operations can be applied to any launch vehicle. This paper presents the mission background as well as results and lessons learned from the flight.

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

    PubMed

    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.

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

    PubMed

    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.

  2. Carbonic Anhydrase Inhibitors. Part 461 Inhibition of Carbonic Anhydrase Isozymes I, II and IV With Trifluoromethylsulfonamide Derivatives and Their Zinc(II) and Copper(II) Complexes

    PubMed Central

    Mincione, Giovanna; Scozzafava, Andrea

    1997-01-01

    Reaction of aromatic/heterocyclic sulfonamides containing a free amino group with triflic anhydride afforded compounds possessing trifluoromethanesulfonamido moieties in their molecule. The Zn(II) and Cu(II) complexes of these new sulfonamides were prepared and characterized by standard procedures (elemental analysis, spectroscopic, magnetic, thermogravimetric and conductimetric measurements). The new derivatives showed good inhibitory activity against three isozymes of carbonic anhydrase (CA), i.e., CA I, II and IV. PMID:18475762

  3. Ares I-X: First Flight of a New Generation

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce R.

    2010-01-01

    The Ares I-X suborbital development flight test demonstrated NASA s ability to design, develop, launch and control a new human-rated launch vehicle (Figure 14). This hands-on missions experience will provide the agency with necessary skills and insights regardless of the future direction of space exploration. The Ares I-X team, having executed a successful launch, will now focus on analyzing the flight data and extracting lessons learned that will be used to support the development of future vehicles.

  4. 75 FR 18245 - Public Federal Regulatory Enforcement Fairness Hearing Region IX Regulatory Fairness Board

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-09

    ... From the Federal Register Online via the Government Publishing Office SMALL BUSINESS ADMINISTRATION Public Federal Regulatory Enforcement Fairness Hearing Region IX Regulatory Fairness Board.... Small Business Administration (SBA) Region IX Regulatory Fairness Board and the SBA Office of...

  5. Docking studies of sulphamate inhibitors of estrone sulphatase in human carbonic anhydrase II.

    PubMed

    Vicker, Nigel; Ho, Yaikat; Robinson, James; Woo, Lawrence L W; Purohit, Atul; Reed, Michael J; Potter, Barry V L

    2003-03-10

    We describe the docking of selected steroidal and non-steroidal estrone sulphatase inhibitors, including the Phase I clinical trial candidate 667COUMATE (6), into the active site of human carbonic anhydrase II (hCA II). The docking scores are compared with the inhibition of hCA II and show good correlation with biological activity.

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

    SciTech Connect

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

    2010-06-23

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

  7. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carbonic anhydrase B and C immunological test system. 866.5200 Section 866.5200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological...

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

    PubMed

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

    2013-11-01

    Extracorporeal CO2 removal from circulating blood is a promising therapeutic modality for the treatment of acute respiratory failure. The enzyme carbonic anhydrase accelerates CO2 removal within gas exchange devices by locally catalyzing HCO3 (-) into gaseous CO2 within the blood. In this work, we covalently immobilized carbonic anhydrase on the surface of polypropylene hollow fiber membranes using glutaraldehyde activated chitosan tethering to amplify the density of reactive amine functional groups for enzyme immobilization. XPS and a colorimetric amine assay confirmed higher amine densities on the chitosan coated fiber compared to control fiber. Chitosan/CA coated fibers exhibited accelerated CO2 removal in scaled-down gas exchange devices in buffer and blood (115% enhancement vs. control, 37% enhancement vs. control, respectively). Carbonic anhydrase immobilized directly on hollow fiber membranes without chitosan tethering resulted in no enhancement in CO2 removal. Additionally, fibers coated with chitosan/carbonic anhydrase demonstrated reduced platelet adhesion when exposed to blood compared to control and heparin coated fibers.

  9. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... system. 866.5200 Section 866.5200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5200 Carbonic anhydrase B and C immunological test system. (a) Identification. A...

  10. 21 CFR 866.5200 - Carbonic anhydrase B and C immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... system. 866.5200 Section 866.5200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5200 Carbonic anhydrase B and C immunological test system. (a) Identification. A...

  11. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  12. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 45 Public Welfare 1 2012-10-01 2012-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  13. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 45 Public Welfare 1 2014-10-01 2014-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  14. 45 CFR 83.5 - Effect of title IX of the Education Amendments of 1972.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 45 Public Welfare 1 2013-10-01 2013-10-01 false Effect of title IX of the Education Amendments of... Purposes; Definitions; Coverage § 83.5 Effect of title IX of the Education Amendments of 1972. The obligations imposed by this part are independent of obligations imposed by or pursuant to title IX of...

  15. Political and Programmatic Impact of Affirmative Action Policy: The Case of Title IX.

    ERIC Educational Resources Information Center

    Bird, Patrick J.

    Title IX legislation has had a widespread impact on institutions of higher education. Similar laws and regulations preceding Title IX include Executive Order 11246, the Comprehensive Health Manpower and Nurse Training Act, and the Equal Employment Opportunity Act. The pervasive influence of Title IX is indicated in its provisions concerning…

  16. 40 CFR Appendix Ix to Part 264 - Ground-Water Monitoring List

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Ground-Water Monitoring List IX... Pt. 264, App. IX Appendix IX to Part 264—Ground-Water Monitoring List Ground-Water Monitoring List... species in the ground water that contain this element are included. 3 CAS index names are those used...

  17. 40 CFR Appendix Ix to Part 264 - Ground-Water Monitoring List

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Ground-Water Monitoring List IX... Pt. 264, App. IX Appendix IX to Part 264—Ground-Water Monitoring List Ground-Water Monitoring List... species in the ground water that contain this element are included. 3 CAS index names are those used...

  18. 40 CFR Appendix Ix to Part 264 - Ground-Water Monitoring List

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 26 2014-07-01 2014-07-01 false Ground-Water Monitoring List IX... Pt. 264, App. IX Appendix IX to Part 264—Ground-Water Monitoring List Ground-Water Monitoring List... species in the ground water that contain this element are included. 3 CAS index names are those used...

  19. 40 CFR Appendix Ix to Part 264 - Ground-Water Monitoring List

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Ground-Water Monitoring List IX... Pt. 264, App. IX Appendix IX to Part 264—Ground-Water Monitoring List Ground-Water Monitoring List... species in the ground water that contain this element are included. 3 CAS index names are those used...

  20. Secondary Athletic Administrators' Perceptions of Title IX Policy Changes

    ERIC Educational Resources Information Center

    Dahl, Gabriel Grawe

    2012-01-01

    The purpose of this study was to investigate North Dakota's Normal Competitive Region (NDNCR) high school athletic administrators' perceptions of 2010 Title IX policy changes respective to their athletic programs. Quantitative and qualitative data were collected to investigate the perceptions. Quantitatively, perception data were gathered from a…

  1. Ares I-X Flight Test Vehicle Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Daniel R.

    2010-01-01

    The first test flight of NASA's Ares I crew launch vehicle, called Ares I-X, was launched on October 28, 2009. Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Flight test data will provide important information on ascent loads, vehicle control, separation, and first stage reentry dynamics. As part of hardware verification, a series of modal tests were designed to verify the dynamic finite element model (FEM) used in loads assessments and flight control evaluations. Based on flight control system studies, the critical modes were the first three free-free bending mode pairs. Since a test of the free-free vehicle was not practical within project constraints, modal tests for several configurations during vehicle stacking were defined to calibrate the FEM. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report describes the test requirements, constraints, pre-test analysis, test execution and results for the Ares I-X flight test vehicle modal test on the Mobile Launcher Platform. Initial comparisons between pre-test predictions and test data are also presented.

  2. Twenty Years after Title IX: Women in Sports Media.

    ERIC Educational Resources Information Center

    Rubarth, Lisa

    1992-01-01

    Though things are better for women in sports media today, Title IX has barely affected their ability to successfully enter into men's sports reporting. The biggest issue is equal access to locker rooms. Women in sports media face less respect, double standards, lack of support, and salary discrepancy. (SM)

  3. National Environmental/Energy Workforce Assessment for Region IX.

    ERIC Educational Resources Information Center

    National Field Research Center Inc., Iowa City, IA.

    This report represents a detailed summation of existing workforce levels, training programs, career potential, and staffing level projections through 1981 for EPA Region IX. This region serves the states of Arizona, California, Hawaii, and Nevada. The specific pollution programs considered include air, noise, pesticides, potable water, radiation…

  4. Implementing Title IX: Concerns of Undergraduate Physical Education Majors.

    ERIC Educational Resources Information Center

    Bain, Linda L.

    A survey of an equal number of male and female students in the University of Houston's secondary school physical education course articulates prospective teacher concerns regarding the implementation of the nondiscriminatory principles set forth in the Title IX ruling. In general, male students evidence greater overall concern about the ruling's…

  5. Ares I-X Malfunction Turn Range Safety Analysis

    NASA Technical Reports Server (NTRS)

    Beaty, J. R.

    2011-01-01

    Ares I-X was the designation given to the flight test version of the Ares I rocket which was developed by NASA (also known as the Crew Launch Vehicle (CLV) component of the Constellation Program). The Ares I-X flight test vehicle achieved a successful flight test on October 28, 2009, from Pad LC-39B at Kennedy Space Center, Florida (KSC). As part of the flight plan approval for the test vehicle, a range safety malfunction turn analysis was performed to support the risk assessment and vehicle destruct criteria development processes. Several vehicle failure scenarios were identified which could have caused the vehicle trajectory to deviate from its normal flight path. The effects of these failures were evaluated with an Ares I-X 6 degrees-of-freedom (6-DOF) digital simulation, using the Program to Optimize Simulated Trajectories Version II (POST2) simulation tool. The Ares I-X simulation analysis provided output files containing vehicle trajectory state information. These were used by other risk assessment and vehicle debris trajectory simulation tools to determine the risk to personnel and facilities in the vicinity of the launch area at KSC, and to develop the vehicle destruct criteria used by the flight test range safety officer in the event of a flight test anomaly of the vehicle. The simulation analysis approach used for this study is described, including descriptions of the failure modes which were considered and the underlying assumptions and ground rules of the study.

  6. Ares I-X Launch Vehicle Modal Test Overview

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Bartolotta, Paul A.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Parks, Russell A.; Lazor, Daniel R.

    2010-01-01

    The first test flight of NASA's Ares I crew launch vehicle, called Ares I-X, is scheduled for launch in 2009. Ares IX will use a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Flight test data will provide important information on ascent loads, vehicle control, separation, and first stage reentry dynamics. As part of hardware verification, a series of modal tests were designed to verify the dynamic finite element model (FEM) used in loads assessments and flight control evaluations. Based on flight control system studies, the critical modes were the first three free-free bending mode pairs. Since a test of the free-free vehicle is not practical within project constraints, modal tests for several configurations in the nominal integration flow were defined to calibrate the FEM. A traceability study by Aerospace Corporation was used to identify the critical modes for the tested configurations. Test configurations included two partial stacks and the full Ares I-X launch vehicle on the Mobile Launcher Platform. This paper provides an overview for companion papers in the Ares I-X Modal Test Session. The requirements flow down, pre-test analysis, constraints and overall test planning are described.

  7. Gender Equity in Intercollegiate Athletics: Determinants of Title IX Compliance

    ERIC Educational Resources Information Center

    Anderson, Deborah J.; Cheslock, John Jesse; Ehrenberg, Ronald G.

    2006-01-01

    Using new data on intercollegiate athletes, this article shows that recent improvement in Title IX compliance among NCAA Division I institutions was previously overestimated, and provides the first estimates of compliance in Divisions II and III. In addition, regression analyses investigate how institutional characteristics relate to the extent of…

  8. N-glycosylations of human α1,3-fucosyltransferase IX are required for full enzyme activity.

    PubMed

    Seelhorst, Katrin; Stacke, Christina; Ziegelmüller, Patrick; Hahn, Ulrich

    2013-05-01

    Human α1,3-fucosyltransferase IX catalyzes the transfer of l-fucose from guanosine diphosphate-β-L-fucose to N-acetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential N-glycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed ∼60% of wt activity and N101Q retained ∼30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.

  9. Ares I-X Flight Data Evaluation: Executive Overview

    NASA Technical Reports Server (NTRS)

    Huebner, Lawrence D.; Waits, David A.; Lewis, Donny L.; Richards, James S.; Coates, R. H., Jr.; Cruit, Wendy D.; Bolte, Elizabeth J.; Bangham, Michal E.; Askins, Bruce R.; Trausch, Ann N.

    2011-01-01

    NASA's Constellation Program (CxP) successfully launched the Ares I-X flight test vehicle on October 28, 2009. The Ares I-X flight was a developmental flight test to demonstrate that this very large, long, and slender vehicle could be controlled successfully. The flight offered a unique opportunity for early engineering data to influence the design and development of the Ares I crew launch vehicle. As the primary customer for flight data from the Ares I-X mission, the Ares Projects Office (APO) established a set of 33 flight evaluation tasks to correlate flight results with prospective design assumptions and models. The flight evaluation tasks used Ares I-X data to partially validate tools and methodologies in technical disciplines that will ultimately influence the design and development of Ares I and future launch vehicles. Included within these tasks were direct comparisons of flight data with preflight predictions and post-flight assessments utilizing models and processes being applied to design and develop Ares I. The benefits of early development flight testing were made evident by results from these flight evaluation tasks. This overview provides summary information from assessment of the Ares I-X flight test data and represents a small subset of the detailed technical results. The Ares Projects Office published a 1,600-plus-page detailed technical report that documents the full set of results. This detailed report is subject to the International Traffic in Arms Regulations (ITAR) and is available in the Ares Projects Office archives files.

  10. Absence or reduction of carbonic anhydrase II in the red cells of the beluga whale and llama: implications for adaptation to hypoxia.

    PubMed

    Yang, H; Hewett-Emmett, D; Tashian, R E

    2000-08-01

    Carbonic anhydrase (CA) expression was examined in the red cells of two mammals that have adapted to low oxygen stress: the llama, which has adapted to high altitudes, and the beluga (or white) whale, which routinely dives for extended periods. Immunodiffusion analyses of their Hb-free hemolysates and partial amino acid sequencing of their HPLC-separated nonheme proteins indicate that the low-activity CA I isozyme is the major nonheme protein in erythrocytes of both the beluga whale and the llama. The high-activity CA II isozyme was not detected in the whale red cells but was present at low levels in erythrocytes of the llama. These results suggest that the absence or decrease in the expression of the high-activity CA II isozyme may be advantageous under hypoxic conditions.

  11. Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii, TweCA.

    PubMed

    Del Prete, Sonia; Vullo, Daniela; De Luca, Viviana; Supuran, Claudiu T; Capasso, Clemente

    2014-12-01

    Diatom genome sequences clearly reveal the presence of different systems for HCO3(-) uptake. Carbon-concentrating mechanisms (CCM) based on HCO3(-) transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO2 hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.

  12. The In Vitro and In Vivo Activity of the Microtubule Disruptor STX140 Is Mediated by Hif-1 Alpha and CAIX Expression.

    PubMed

    Stengel, Chloe; Newman, Simon P; Leese, Mathew P; Thomas, Mark P; Potter, Barry V L; Reed, Michael J; Purohit, Atul; Foster, Paul A

    2015-10-01

    Tumor neo-angiogenesis is regulated, in part, by the hypoxia-inducible gene HIF1. Evidence suggests HIF1 associates with polymerized microtubules and traffics to the nucleus. This study investigated the role of HIF1 in mediating the antitumor activity of two steroid-based sulfamate ester microtubule disruptors, STX140 and STX243, in vitro and in vivo. The effects of STX140, STX243 and the parental compound 2-methoxyestradiol (STX66) on HIF1α and HIF2α protein expression were assessed in vitro in MCF-7 and MDA-MB-231 cells cultured under hypoxia. More pertinently, their effects were examined on HIF1-regulated genes in vivo in mice bearing MCF-7 or MDA-MB-231 tumors. The level of mRNA expression of vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUTI), phosphoglycerate kinase (PGK), ATP-binding cassette sub-family B member 1 (ABCB1) and carbonic anhydrase IX (CAIX) was quantified by Real-time Polymerase Chain Reaction (RT-PCR). Despite inhibiting nuclear HIF1α protein accumulation under hypoxia in vitro, STX140 and STX243 did not significantly regulate the expression of four out of five HIF1α-regulated genes in vitro and in vivo. Only CAIX mRNA expression was down-regulated both in vitro and in vivo. Immunoblot analysis showed that STX140 and STX243 reduced CAIX protein expression in vitro. These compounds had no effect on HIF2α translocation. The potential for inhibition of CAIX by STX140 and STX243 was examined by docking the ligands to the active site in comparison with a known sulfamate-based inhibitor. Microtubule disruption and antitumor activity of STX140 and STX243 is most likely HIF1-independent and may, at least in part, be mediated by inhibition of CAIX expression and activity.

  13. 40 CFR Appendix Ix to Part 268 - Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B)

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B) IX Appendix IX to Part 268 Protection of.... 268, App. IX Appendix IX to Part 268—Extraction Procedure (EP) Toxicity Test Method and...

  14. 40 CFR Appendix Ix to Part 268 - Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B)

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 27 2011-07-01 2011-07-01 false Extraction Procedure (EP) Toxicity Test Method and Structural Integrity Test (Method 1310B) IX Appendix IX to Part 268 Protection of.... 268, App. IX Appendix IX to Part 268—Extraction Procedure (EP) Toxicity Test Method and...

  15. The Development of the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    Ess, Robert H.

    2008-01-01

    The National Aeronautics and Space Administration (NASA) Constellation Program (CxP) has identified a series of tests to provide insight into the design and development of the Ares I Crew Launch Vehicle (CLV) and the Orion Crew Exploration Vehicle (CEV). Ares I-X was created as the first suborbital development flight test to help meet CxP objectives. The Ares I-X flight vehicle is an early operational model of Ares, with specific emphasis on Ares I and ground operation characteristics necessary to meet Ares I-X flight test objectives. Ares I-X will encompass the design and construction of an entire system that includes the Flight Test Vehicle (FTV) and associated operations. The FTV will be a test model based on the Ares I design. Select design features will be incorporated in the FTV design to emulate the operation of the CLV in order to meet the flight test objectives. The operations infrastructure and processes will be customized for Ares I-X, while still providing data to inform the developers of the launch processing system for Ares/Orion. The FTV is comprised of multiple elements and components that will be developed at different locations. The components will be delivered to the launch/assembly site, Kennedy Space Center (KSC), for assembly of the elements and components into an integrated, flight-ready, launch vehicle. The FTV will fly a prescribed trajectory in order to obtain the necessary data to meet the objectives. Ares I-X will not be commanded or controlled from the ground during flight, but the FTV will be equipped with telemetry systems, a data recording capability and a flight termination system (FTS). The in-flight part of the test includes a trajectory to simulate maximum dynamic pressure during flight and perform a stage separation representative of the CLV. The in-flight test also includes separation of the Upper Stage Simulator (USS) from the First Stage and recovery of the First Stage. The data retrieved from the flight test will be analyzed

  16. In-vivo fluorescence dosimetry of aminolevulinate-based protoporphyrin IX (PpIX) accumulation in human nonmelanoma skin cancers and precancers

    NASA Astrophysics Data System (ADS)

    Warren, Christine B.; Lohser, Sara; Chang, Sung; Bailin, Philip A.; Maytin, Edward V.

    2009-06-01

    PDT is clinically useful for precancers (actinic keratoses; AK) of the skin, but the optimal duration for 5-ALA application is still controversial. For basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), cure rates remain inferior to surgical excision. Lack of knowledge about regional levels of PpIX levels within target tissues clearly contribute to these suboptimal results. To investigate PpIX levels achievable in human skin neoplasias in-vivo, a clinical study to monitor PpIX accumulation in vivo was performed. PpIX-fluorescence in patients undergoing ALA-PDT for facial AK was monitored via real-time in-vivo fluorescence dosimetry, with measurements q20 min following application of 5-ALA (Levulan Kerastick). PpIX accumulation followed linear kinetics in nearly all cases. The slopes varied widely, and did not correlate with clinical outcome in all patients. Some patients with a low accumulation of PpIX fluorescence had a good response to therapy, whereas others with high PpIX accumulation required repeat treatment (although not necessarily of the same lesion). PpIX accumulation rates did correlate to a certain degree with the overall amount of erythema. We conclude that unknown factors besides PpIX levels must be critical for the response to treatment. To assess the relationship between PpIX levels in various skin cancers, patients undergoing routine Mohs surgery for BCC or SCC were measured by in-vivo dosimetry at 2 h after 5-ALA application. Overall, a progressive increase in PpIX signal during malignant progression was observed, in the following rank order: Normal skin < AK < SCC ~ BCC.

  17. An investigation of carbonic anhydrase activity in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate (Raja rhina), and spotted raffish (Hydrolagus colliei).

    PubMed

    Gilmour, Kathleen M; Shah, Bina; Szebedinszky, Cheryl

    2002-01-01

    Separated plasma and whole blood non-bicarbonate buffering capacities, together with plasma and gill carbonic anhydrase activities and endogenous plasma carbonic anhydrase inhibitor activity were investigated in three species of fish: the brown bullhead (Ameirus nebulosus), a teleost; the longnose skate (Raja rhina), an elasmobranch; and the spotted ratfish (Hydrolagus colliei), a chimaeran. The objective was to test the hypothesis that species possessing gill membrane-bound carbonic anhydrase and/or plasma carbonic anhydrase activity would also exhibit high plasma nonbicarbonate buffering capacity relative to whole blood non-bicarbonate buffering capacity and would lack an endogenous plasma carbonic anhydrase inhibitor. Separated plasma non-bicarbonate buffering capacity constituted > or = 40% of whole-blood buffering in all three species. In addition, all species lacked an endogenous plasma carbonic anhydrase inhibitor. Separated plasma from skate and ratfish contained carbonic anhydrase activity, whereas bullhead plasma did not. Examination of the subcellular distribution and characteristics of branchial carbonic anhydrase activity revealed that the majority of branchial carbonic anhydrase activity originated from the cytoplasmic fraction in all species, with only 3-5% being associated with a microsomal fraction. The microsomal carbonic anhydrase activity of bullhead and ratfish was significantly reduced by washing, indicating the presence of carbonic anhydrase activity that was not integrally associated with the membrane pellet, microsomal carbonic anhydrase activity in skate was unaffected by washing. In addition, microsomal carbonic anhydrase activity from skate and ratfish but not bullhead gills was released to a significant extent from its membrane association by treatment with phosphatidylinositol-specific phospholipase C. The results obtained for skate are consistent with published data for dogfish, suggesting that the possession of branchial membrane

  18. Binding determinants of the small heat shock protein, αB-crystallin: recognition of the ‘IxI' motif

    PubMed Central

    Delbecq, Scott P; Jehle, Stefan; Klevit, Rachel

    2012-01-01

    Small heat shock proteins (sHSPs) play a central role in protein homeostasis under conditions of stress by binding partly unfolded, aggregate-prone proteins and keeping them soluble. Like many sHSPs, the widely expressed human sHSP, αB-crystallin (‘αB'), forms large polydisperse multimeric assemblies. Molecular interactions involved in both sHSP function and oligomer formation remain to be delineated. A growing database of structural information reveals that a central conserved α-crystallin domain (ACD) forms dimeric building blocks, while flanking N- and C-termini direct the formation of larger sHSP oligomers. The most commonly observed inter-subunit interaction involves a highly conserved C-terminal ‘IxI/V' motif and a groove in the ACD that is also implicated in client binding. To investigate the inherent properties of this interaction, peptides mimicking the IxI/V motif of αB and other human sHSPs were tested for binding to dimeric αB-ACD. IxI-mimicking peptides bind the isolated ACD at 22°C in a manner similar to interactions observed in the oligomer at low temperature, confirming these interactions are likely to exist in functional αB oligomers. PMID:23188086

  19. Aggressive therapy improves cirrhosis in glycogen storage disease type IX.

    PubMed

    Tsilianidis, Laurie A; Fiske, Laurie M; Siegel, Sara; Lumpkin, Chris; Hoyt, Kate; Wasserstein, Melissa; Weinstein, David A

    2013-06-01

    Glycogen storage disease type IX (GSD IX) is described as a benign condition that often does not require treatment. Most patients with the disease are thought to outgrow the childhood manifestations, which include hepatomegaly, poor growth, and ketosis with or without hypoglycemia. Long term complications including fibrosis and cirrhosis have seldom been reported in the most common subtype, GSD IXα. We present two cases of children with GSD IXα who had fibrosis at the time of diagnosis in addition to the commonly reported disease manifestations. Structured therapy with frequent doses of uncooked cornstarch and protein supplementation was initiated, and both children responded with improved growth velocity, increased energy, decreased hepatomegaly and improved well-being. Additionally, radiographic features of fibrosis improved. We propose that GSD IXα is not a benign condition. Even in patients with a less severe presentation, consideration of a structured treatment regimen to improve quality of life appears warranted.

  20. Bianchi-IX, Darboux-Halphen and Chazy-Ramanujan

    NASA Astrophysics Data System (ADS)

    Chanda, Sumanto; Guha, Partha; Roychowdhury, Raju

    2016-02-01

    Bianchi-IX four metrics are SU(2) invariant solutions of vacuum Einstein equation, for which the connection-wise self-dual case describes the Euler top, while the curvature-wise self-dual case yields the Ricci flat classical Darboux-Halphen system. It is possible to see such a solution exhibiting Ricci flow. The classical Darboux-Halphen system is a special case of the generalized one that arises from a reduction of the self-dual Yang-Mills equation and the solutions to the related homogeneous quadratic differential equations provide the desired metric. A few integrable and near-integrable dynamical systems related to the Darboux-Halphen system and occurring in the study of Bianchi-IX gravitational instanton have been listed as well. We explore in details whether self-duality implies integrability.

  1. Ares I-X Best Estimated Trajectory Analysis and Results

    NASA Technical Reports Server (NTRS)

    Karlgaard, Christopher D.; Beck, Roger E.; Starr, Brett R.; Derry, Stephen D.; Brandon, Jay; Olds, Aaron D.

    2011-01-01

    The Ares I-X trajectory reconstruction produced best estimated trajectories of the flight test vehicle ascent through stage separation, and of the first and upper stage entries after separation. The trajectory reconstruction process combines on-board, ground-based, and atmospheric measurements to produce the trajectory estimates. The Ares I-X vehicle had a number of on-board and ground based sensors that were available, including inertial measurement units, radar, air-data, and weather balloons. However, due to problems with calibrations and/or data, not all of the sensor data were used. The trajectory estimate was generated using an Iterative Extended Kalman Filter algorithm, which is an industry standard processing algorithm for filtering and estimation applications. This paper describes the methodology and results of the trajectory reconstruction process, including flight data preprocessing and input uncertainties, trajectory estimation algorithms, output transformations, and comparisons with preflight predictions.

  2. Ares I-X Thermal Model Correlation and Lessons Learned

    NASA Technical Reports Server (NTRS)

    Amundsen, Ruth M.

    2010-01-01

    The Ares I-X vehicle launched and flew successfully on October 28, 2009. This paper will describe the correlation of the vehicle thermal model to both ground testing and flight data. A main purpose of the vehicle model and ground testing was to ensure that the avionics within the vehicle were held within their thermal limits prior to launch and during flight. The correlation of the avionics box temperatures will be shown. Also, the lessons learned in the thermal discipline during the modeling, test, correlation to test, and flight of the Ares I-X flight test vehicle will be described. Lessons learned will cover thermal modeling, as well as management of the thermal discipline, thermal team, and thermal-related actions in design, testing, and flight.

  3. Hantavirus Prevalence in the IX Region of Chile

    PubMed Central

    Vial, Pablo C.; Castillo, Constanza H.; Godoy, Paula M.; Hjelle, Brian; Ferrés, Marcela G.

    2003-01-01

    An epidemiologic and seroprevalence survey was conducted (n=830) to assess proportion of persons exposed to hantavirus in IX Region Chile, which accounts for 25% of reported cases of hantavirus cardiopulmonary syndrome. This region has three geographic areas with different disease incidences and a high proportion of aboriginals. Serum samples were tested for immunoglobulin (Ig) G antibodies by enzyme-linked immunosorbent assay against Sin Nombre virus N antigen by strip immunoblot assay against Sin Nombre, Puumala, Río Mamoré, and Seoul N antigens. Samples from six patients were positive for IgG antibodies reactive with Andes virus; all patients lived in the Andes Mountains. Foresting was also associated with seropositivity; but not sex, age, race, rodent exposure, or farming activities. Exposure to hantavirus varies in different communities of IX Region. Absence of history of pneumonia or hospital admission in persons with specific IgG antibodies suggests that infection is clinically inapparent. PMID:12890323

  4. Sulfa drugs as inhibitors of carbonic anhydrase: new targets for the old drugs.

    PubMed

    al-Rashida, Mariya; Hussain, Sajad; Hamayoun, Mehwish; Altaf, Aisha; Iqbal, Jamshed

    2014-01-01

    Sulfa drugs are well-known antibacterial agents containing N-substituted sulfonamide group on para position of aniline ring (NH2RSO2NHR'). In this study 2,4-dichloro-1,3,5-triazine derivatives of sulfa drugs, sulfamerazine (1b), sulfaquinoxaline (2b), sulfadiazine (3b), sulfadimidine (4b), and sulfachloropyrazine (5b) (1a-5a) were synthesized and characterized. Their carbonic anhydrase inhibition activity was evaluated against bovine cytosolic carbonic anhydrase isozyme II (bCA II). For the sake of comparison the CA inhibition activity of the parent sulfa drugs (1b-5b) was also evaluated. A significant increase in CA inhibition activity of sulfa drugs was observed upon substitution with 2,4-dichloro-1,3,5-triazine moiety. Molecular docking studies were carried out to highlight binding site interactions. ADME properties were calculated to evaluate drug likeness of the compounds.

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

    PubMed

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

    2000-01-01

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

  6. Discovery of Potent Carbonic Anhydrase and Acetylcholinesterase Inhibitors: 2-Aminoindan β-Lactam Derivatives

    PubMed Central

    Genç, Hayriye; Kalin, Ramazan; Köksal, Zeynep; Sadeghian, Nastaran; Kocyigit, Umit M.; Zengin, Mustafa; Gülçin, İlhami; Özdemir, Hasan

    2016-01-01

    β-Lactams are pharmacologically important compounds because of their various biological uses, including antibiotic and so on. β-Lactams were synthesized from benzylidene-inden derivatives and acetoxyacetyl chloride. The inhibitory effect of these compounds was examined for human carbonic anhydrase I and II (hCA I, and II) and acetylcholinesterase (AChE). The results reveal that β-lactams are inhibitors of hCA I, II and AChE. The Ki values of β-lactams (2a–k) were 0.44–6.29 nM against hCA I, 0.93–8.34 nM against hCA II, and 0.25–1.13 nM against AChE. Our findings indicate that β-lactams (2a–k) inhibit both carbonic anhydrases (CA) isoenzymes and AChE at low nanomolar concentrations. PMID:27775608

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

    PubMed

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

    2016-11-01

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

  8. Loop quantum cosmology of Bianchi type IX models

    NASA Astrophysics Data System (ADS)

    Wilson-Ewing, Edward

    2010-08-01

    The loop quantum cosmology “improved dynamics” of the Bianchi type IX model are studied. The action of the Hamiltonian constraint operator is obtained via techniques developed for the Bianchi type I and type II models, no new input is required. It is shown that the big bang and big crunch singularities are resolved by quantum gravity effects. We also present effective equations which provide quantum geometry corrections to the classical equations of motion.

  9. Loop quantum cosmology of Bianchi type IX models

    SciTech Connect

    Wilson-Ewing, Edward

    2010-08-15

    The loop quantum cosmology 'improved dynamics' of the Bianchi type IX model are studied. The action of the Hamiltonian constraint operator is obtained via techniques developed for the Bianchi type I and type II models, no new input is required. It is shown that the big bang and big crunch singularities are resolved by quantum gravity effects. We also present effective equations which provide quantum geometry corrections to the classical equations of motion.

  10. Ares I-X: On the Threshold of Exploration

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce

    2009-01-01

    Ares I-X, the first flight of the Ares I crew launch vehicle, is less than a year from launch. Ares I-X will test the flight characteristics of Ares I from liftoff to first stage separation and recovery. The flight also will demonstrate the computer hardware and software (avionics) needed to control the vehicle; deploy the parachutes that allow the first stage booster to land in the ocean safely; measure and control how much the rocket rolls during flight; test and measure the effects of first stage separation; and develop and try out new ground handling and rocket stacking procedures in the Vehicle Assembly Building (VAB) and first stage recovery procedures at Kennedy Space Center (KSC) in Florida. All Ares I-X major elements have completed their critical design reviews, and are nearing final fabrication. The first stage--four-segment solid rocket booster from the Space Shuttle inventory--incorporates new simulated forward structures to match the Ares I five-segment booster. The upper stage, Orion crew module, and launch abort system will comprise simulator hardware that incorporates developmental flight instrumentation for essential data collection during the mission. The upper stage simulator consists of smaller cylindrical segments, which were transported to KSC in fall 2008. The crew module and launch abort system simulator were shipped in December 2008. The first stage hardware, active roll control system (RoCS), and avionics components will be delivered to KSC in 2009. This paper will provide detailed statuses of the Ares I-X hardware elements as NASA's Constellation Program prepares for this first flight of a new exploration era in the summer of 2009.

  11. Technical Progress on the Ares I-X Flight Test

    NASA Technical Reports Server (NTRS)

    Davis, S.R.; Robinson, K.F.; Flynn, K.C.

    2008-01-01

    Ares I-X will be NASA's first test flight for a new human-rated launch vehicle since 1981, and the team is well on its way toward completing the vehicle's design and hardware fabrication for an April 2009 launch. This uncrewed suborbital development test flight gives NASA its first opportunities to: gather critical data about the flight dynamics of the integrated launch vehicle; understand how to control its roll during flight; better characterize the stage separation environments during future flight; and demonstrate the first stage recovery system. The Ares I-X Flight Test Vehicle (FTV) incorporates a mix of flight and mockup hardware. It is powered by a four-segment solid rocket booster, and will be modified to include a fifth, spacer segment; the upper stage, Orion crew exploration vehicle, and launch abort system are simulator hardware to make the FTV aerodynamically similar to the same size, shape, and weight of Ares I. The Ares IX first stage includes an existing Shuttle solid rocket motor and thrust vector control system controlled by an Ascent Thrust Vector Controller (ATVC) designed and built by Honeywell International. The avionics system will be tested in a dedicated System Integration Laboratory located at Lockheed Martin Space Systems (LMSS) in Denver, Colorado. The Upper Stage Simulator (USS) is made up of cylindrical segments that will be stacked and integrated at Kennedy Space Center (KSC) for launch. Glenn Research Center is already building these segments, along with their internal access structures. The active Roll Control System (RoCS) includes two thruster units harvested from Peacekeeper missiles. Duty cycle testing for RoCS was conducted, and fuel tanking and detanking tests will occur at KSC in early 2008. This important flight will provide valuable experience for the ground operations team in integrating, stacking, and launching Ares I. Data from Ares I-X will ensure the safety and reliability of America's newest launch vehicle.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    SciTech Connect

    Weiwei, Zhang; Hu, Renming

    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.

  14. Optimization and characterization of the endogenous production of protoporphyrin IX in a yeast model

    NASA Astrophysics Data System (ADS)

    Joniová, Jaroslava; Gerelli, Emmanuel; Zellweger, Matthieu; Wagnières, Georges

    2016-12-01

    The availability of reproducible, convenient, and inexpensive model organisms able to generate predictable levels of endogenous porphyrins, including protoporphyrin IX (PpIX), is essential in photomedicine research. Saccharomyces cerevisiae produces endogenous PpIX and was used as a model organism for this study with the aim to maximize endogenous PpIX fluorescence intensity. It was found that PpIX fluorescence was significantly enhanced by administration of 5-aminolevulinic acid (ALA) and 2,2‧-bipyridyl. Fluorescence intensity and spectroscopy of PpIX produced endogenously were measured in diluted yeast solutions under various conditions. The optimal protocol was: 5 μM ALA and 1 mM 2,2‧-bipyridyl administered synchronously at 32°C. After 3 h, PpIX in yeast demonstrated similar steady-state and time-resolved spectroscopy as that of PpIX in DMSO. Moreover, under hypoxic conditions, the reciprocal lifetime of PpIX delayed fluorescence measured in real time was correlated to the partial pressure of oxygen (pO2) measured concomitantly with a commercially available pO2 probe. These data show that yeast can, in optimal conditions, reproducibly generate PpIX. This is of interest in various fields such as photodiagnosis, photodynamic therapy, and photobiomodulation. Use of this model organism focuses on essential mechanisms, without the complexity of a multicellular organism.

  15. New polymorphic variants of human blood clotting factor IX

    SciTech Connect

    Surin, V.L.; Luk`yanenko, A.V.; Tagiev, A.F.; Smirnova, O.V.; Plutalov, O.V.; Berlin, Yu.A.

    1995-04-01

    The polymorphism of Alu-repeats, which are located in the introns of the human factor IX gene (copies 1-3), was studied. To identify polymorphic variants, direct sequencing of PCR products that contained appropriate repeats was used. In each case, 20 unrelated X chromosomes were studied. A polymorphic Dra I site was found near the 3{prime}-end of Alu copy 3 within the region of the polyA tract. A PCR-based testing system with internal control of restriction hydrolysis was suggested. Testing 81 unrelated X chromosomes revealed that the frequency of the polymorphic Dra I site is 0.23. Taq I polymorphism, which was revealed in Alu copy 4 of factor IX gene in our previous work, was found to be closely linked to Dra I polymorphism. Studies in linkage between different types of polymorphisms of the factor IX gene revealed the presence of a rare polymorphism in intron a that was located within the same minisatellite region as the known polymorphic insertion 50 bp/Dde I. However, the size of the insertion in our case was 26 bp. Only one polymorphic variant was found among over 150 unrelated X chromosomes derived from humans from Moscow and its vicinity. 10 refs., 4 figs., 1 tab.

  16. Bladder tissue diagnostics utilizing Protoporphyrin IX fluorescence detection

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Rick, Kai; Steinbach, Pia; Kriegmair, M.

    1995-01-01

    Instillation of a solution of 5-aminolevulinic acid (5-ALA) into the urinary bladder leads to a tumorselective accumulation of fluorescing Protoporphyrin IX (PpIX) within hours. Upon fluorescence excitation using a Kr+- laser, cystoscopy provides high contrast images even of early stage tumors, that are invisible or hardly detectable by routine white light cystoscopy. Fluorescence can simply be judged by naked eyes or recorded with a target integrating camera in real color. Histological and fluorescence data of 91 patients were evaluated statistically, showing a sensitivity of 97% and a specificity of 68% for the detection of dysplastic lesions or malignant tumors. The detectability of a sufficient fluorescence contrast of suspicious versus normal tissue is not affected significantly by either short incubation times of less than 1 hour or prolonged retention times without 5-ALA in the instillation liquid of up to about 6 hours. The fluorescence intensity detected from the tissue surface is not only dependent on PpIX concentration. The additional influence of optical parameters of tissue and fluorochrome distribution on the fluorescence signal was determined using Monte Carlo computer simulations. Results show that 5-ALA induced fluorochrome detection is superior to the detection of fluorochromes that do not exclusively stain the epithelium. Using the ratio of fluorescence intensity to backscattered excitation light corrects for geometrical and absorption effects but would introduce a dependence on the scattering coefficient.

  17. Loop quantum cosmology of Bianchi IX: effective dynamics

    NASA Astrophysics Data System (ADS)

    Corichi, Alejandro; Montoya, Edison

    2017-03-01

    We study solutions to the effective equations for the Bianchi IX class of spacetimes within loop quantum cosmology (LQC). We consider Bianchi IX models whose matter content is a massless scalar field, by numerically solving the loop quantum cosmology effective equations, with and without inverse triad corrections. The solutions are classified using certain geometrically motivated classical observables. We show that both effective theories—with lapse N  =  V and N  =  1—resolve the big bang singularity and reproduce the classical dynamics far from the bounce. Moreover, due to the positive spatial curvature, there is an infinite number of bounces and recollapses. We study the limit of large field momentum and show that both effective theories reproduce the same dynamics, thus recovering general relativity. We implement a procedure to identify amongst the Bianchi IX solutions, those that behave like k  =  0,1 FLRW as well as Bianchi I, II, and VII0 models. The effective solutions exhibit Bianchi I phases with Bianchi II transitions and also Bianchi VII0 phases, which had not been studied before. We comment on the possible implications of these results for a quantum modification to the classical BKL behaviour.

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

    PubMed

    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.

  19. The role of carbonic anhydrase VI in bitter taste perception: evidence from the Car6−/− mouse model

    PubMed Central

    2014-01-01

    Background Carbonic anhydrase VI (CA VI) is a secretory isozyme of the α-CA gene family. It is highly expressed in the salivary and mammary glands and secreted into saliva and milk. Although CA VI was first described as a gustatory protein, its exact functional roles have remained enigmatic. Interestingly, polymorphism of the CA6 gene was recently linked to bitter taste perception in humans. In this study, we compared the preference of Car6−/− and wild-type mice for different taste modalities in an IntelliCage monitoring environment. Morphologies of taste buds, tongue papillae, and von Ebner’s glands were evaluated by light microscopy. Cell proliferation and rate of apoptosis in tongue specimens were examined by Ki67 immunostaining and fluorescent DNA fragmentation staining, respectively. Results The behavioral follow up of the mice in an IntelliCage system revealed that Car6−/− mice preferred 3 μM quinine (bitter) solution, whereas wild type mice preferred water. When the quinine concentration increased, both groups preferentially selected water. Histological analysis, Ki67 immunostaining and detection of apoptosis did not reveal any significant changes between tongue specimens of the knockout and wild type mice. Conclusions Our knockout mouse model confirms that CA VI is involved in bitter taste perception. CA VI may be one of the factors which contribute to avoidance of bitter, potentially harmful, substances. PMID:25134447

  20. Transport Activity of the Sodium Bicarbonate Cotransporter NBCe1 Is Enhanced by Different Isoforms of Carbonic Anhydrase

    PubMed Central

    Schueler, Christina; Becker, Holger M.; McKenna, Robert; Deitmer, Joachim W.

    2011-01-01

    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. PMID:22076132

  1. Activity and anion inhibition studies of the α-carbonic anhydrase from Thiomicrospira crunogena XCL-2 Gammaproteobacterium.

    PubMed

    Mahon, Brian P; Díaz-Torres, Natalia A; Pinard, Melissa A; Tu, Chingkuang; Silverman, David N; Scott, Kathleen M; McKenna, Robert

    2015-11-01

    Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs (k(cat)/K(M)=1.1×10(7) M(-1) s(-1)), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl(-), Br(-), and I(-), all showing Ki values in the millimolar range (53-361 mM). Hydrogen sulfide (HS(-)) revealed the highest affinity for TcruCA with a Ki of 1.1 μM. It is predicted that inhibition of TcruCA by HS(-) (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism's metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena.

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

    PubMed Central

    Modakh, Joyanta K.; Liu, Yu C.; Machuca, Mayra A.; Supuran, Claudiu T.; Roujeinikova, Anna

    2015-01-01

    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. PMID:26010545

  3. Specific arginine modification at the phosphatase site of muscle carbonic anhydrase.

    PubMed

    Pullan, L M; Noltmann, E A

    1985-01-29

    Mammalian carbonic anhydrase III has previously been shown to catalyze the hydrolysis of p-nitrophenyl phosphate in addition to possessing the conventional CO2 hydratase and p-nitrophenylacetate esterase activities. Modification of pig muscle carbonic anhydrase III with the arginine reagent phenylglyoxal yielded two clearly distinctive results. Reaction of the enzyme with phenylglyoxal at concentrations equivalent to those of the enzyme yielded stoichiometric inactivation titration of the enzyme's phosphatase activity, approaching 100% loss of activity with the simultaneous modification of one arginine residue, the latter based on a 1:1 reaction of phenylglyoxal with arginine. At this low ratio of phenylglyoxal to enzyme, neither the CO2 hydratase activity nor the acetate esterase activity was affected. When the modification was performed with a significant excess of phenylglyoxal, CO2 hydratase and acetate esterase activities were diminished as well. That loss of activity was accompanied by the incorporation of an additional half dozen phenylglyoxals and, presumably, the modification of an equal number of arginine residues. The data in their entirety are interpreted to show that the p-nitrophenylphosphatase activity is a unique property of carbonic anhydrase III and that excessive amounts of the arginine-modifying reagent lead to unspecific structural changes of the enzyme as a result of which all of its enzymatic activities are inactivated.

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

    SciTech Connect

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

    2008-01-01

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

  5. Characterization and inhibition studies of carbonic anhydrase from gill of Russian Sturgeon Fish (Acipenser gueldenstaedtii).

    PubMed

    Dinçer, Barbaros; Ekinci, Arife Pınar; Akyüz, Gülay; Kurtoğlu, İlker Zeki

    2016-12-01

    An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from gill of Acipenser gueldenstaedtii as an endangered sturgeon species. The carbonic anhydrase was purified 66-folds with yield 20.7% by Sepharose-4B-l-tyrosine-sulfanilamide affinity column and the specific activity was determined as 222.2 EU/mg protein. Km and Vmax kinetic values for gill carbonic anhydrase were calculated by a Lineweaver-Burk graph using p-nitrophenol acetate (p-NPA) as a substrate, and was defined as 2.5 mM and 5 × 10(6 )μM/min, respectively. It was observed that CA from the sturgeon gill in the presence of the sulfanilamide and acetazolamide as an inhibitor had very low IC50 values such as 13.0 and 0.1 μM, respectively. In addition, it was determined that the enzyme was inhibited by Fe(2+,) Co(2+,) Ni(2+), and Zn(2+)-Ba(2+) with the IC50 values of 0.2, 1.7, 1.2, and 1.1 mM, respectively.

  6. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    PubMed

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  7. Targeting of p53 and its homolog p73 by protoporphyrin IX.

    PubMed

    Sznarkowska, Alicja; Maleńczyk, Katarzyna; Kadziński, Leszek; Bielawski, Krzysztof P; Banecki, Bogdan; Zawacka-Pankau, Joanna

    2011-01-03

    The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.

  8. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation

    PubMed Central

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; De Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-01-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation. PMID:14528266

  9. Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3

    PubMed Central

    Krishnan, Devishree; Liu, Lei; Wiebe, Shane A.; Casey, Joseph R.; Cordat, Emmanuelle; Alexander, R. Todd

    2016-01-01

    Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter’s activity. PMID:26041446

  10. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Adoption of section IX of the ASME Code. 57.02-2 Section... AND BRAZING General Requirements § 57.02-2 Adoption of section IX of the ASME Code. (a) The... in this part. Table 57.02-1(a)—Limitations and Modifications to the Adoption of section IX of...

  11. 46 CFR 57.02-2 - Adoption of section IX of the ASME Code.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Adoption of section IX of the ASME Code. 57.02-2 Section... AND BRAZING General Requirements § 57.02-2 Adoption of section IX of the ASME Code. (a) The... in this part. Table 57.02-1(a)—Limitations and Modifications to the Adoption of section IX of...

  12. The spectroscopy analyses of PpIX by ultrasound irradiation and its sonotoxicity in vitro.

    PubMed

    Wang, Pan; Wang, Xiaobing; Zhang, Kun; Gao, Kaili; Song, Ming; Liu, Quanhong

    2013-07-01

    Protoporphyrin IX (PpIX) has been used as a sensitizer in photodynamic therapy (PDT) as well as in sonodynamic therapy (SDT). The photo-bleaching of PpIX has been well investigated in many experimental systems and some photo-products have also been identified in PDT. But until now, little information has been reported about the sono-damage of PpIX in SDT. So, the present study was to investigate changes of PpIX properties before and after different ultrasound treatment, and the potential interactions between PpIX, ultrasound and the irradiated cells. In cell-free system, the absorption and fluorescence spectra of PpIX in different solutions were measured by ultraviolet spectrometer and fluorescence spectrophotometer, respectively. The terephthalic acid dosimetry was applied to evaluate the efficiency of ultrasound cavitation by monitoring hydroxyl radical (OH) production on the thermolysis of H2O in the ultrasound field. In in vitro study, confocal microscopy was applied to detect the sub-cellular localization of PpIX in S180 cells before and after ultrasound exposure. Flow cytometry was used to detect the reactive oxygen species (ROS) generation during PpIX-SDT. MTT assay was performed to evaluate the cell viability of S180 cells after SDT treatment with or without ROS scavengers. The results show that PpIX displayed different spectral patterns in different solutions. PpIX was decomposed by ultrasound exposure as measured by the decreased absorption and fluorescence peak values in RPMI-1640 medium. In addition, the decomposition of PpIX was found to be simultaneously accompanied by OH production with increasing output power from ultrasound generator. PpIX at 1μg/ml significantly enhanced the ultrasound induced cavitation as measured by OH generation, and which was greatly eliminated by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD. The in vitro study indicates more PpIX entered into S180 cells after ultrasound exposure. And, the extra-cellular PpIX

  13. Cell cycle dependence of protophorphyrin IX generation in 9L rat gliosarcoma

    NASA Astrophysics Data System (ADS)

    Luo, Shiming; Da, Xing; Chen, Qun

    2006-09-01

    Photodynamic therapy (PDT) is a cancer therapy that utilizes optical energy to activate a photosensitizer drug in a target tissue. Always, the curative effect is dependent on the light fluence, the concentration of the photosensitizer and the concentration of the oxygen. To date, Protophorphyrin IX (PpIX) as the only one endogenous photosensitizer is widely used in PDT of brain tumors. Since PpIX is synthesized in intracellular structure, and is likely dependent on the phase of the cell cycle. The cell cycle dependence of PpIX production is thus investigated in the current work in 9L gliosarcoma cells.

  14. The endothelial cell binding determinant of human factor IX resides in the. gamma. -carboxyglutamic acid domain

    SciTech Connect

    Toomey, J.R.; Roberts, H.R.; Stafford, D.W. ); Smith, K.J. United Blood Services, Albuquerque, NM )

    1992-02-18

    The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a K{sub d} of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the {gamma}-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, the authors performed competitive binding experiments between {sup 125}I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. The data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.

  15. Evaluation of PpIX formation in Cervical Intraepithelial Neoplasia I (CIN) using widefield fluorescence images

    NASA Astrophysics Data System (ADS)

    Carbinatto, Fernanda M.; Inada, Natalia M.; Fortunato, Thereza C.; Lombardi, Welington; da Silva, Eduardo V.; Vollet Filho, José D.; Kurachi, Cristina; Pratavieira, Sebastião.; Bagnato, Vanderlei S.

    2016-03-01

    Optical techniques has been described as auxiliary technology for screening of neoplasia because shows the potential for tissues differentiation in real-time and it is a noninvasive detection and safe. However, only endogenous fluorophores presents the lesion may be insufficient and needed of the administration of the fluorophores synthesized, such as, precursor molecule of protoporphyrin IX (PpIX) induced by 5- aminolevulinic acid and your derivatives. Topical application of methylaminolevulinate (MAL), induces formation of the endogenous photosensitizer, PpIX in tissues where carcinogenesis has begun. The PpIX tend to accumulate in premalignant and malignant tissues and the illumination with light with appropriate wavelength beginning to excitation of PpIX fluorescence, which helps to localize PpIX-rich areas and identify potentially malignant tissues. The aim of the study is to evaluate the production of PpIX in the cervix with CIN I through of the fluorescence images captured after 1 hour of cream application. It was possible to visualize PpIX fluorescence in cervix and it was possible to observe the selectivity in fluorescence in squamous-columnar junction, which a pre-cancerous condition (CIN) and usually is localized. Through the image processing it was possible to quantify the increase of red fluorescence. For the CIN I the increase of red fluorescence was approximately of 4 times indicating a good PpIX formation.

  16. Noninvasive murine glioma detection improved following photobleaching of skin PpIX fluorescence

    NASA Astrophysics Data System (ADS)

    Gibbs-Strauss, Summer L.; Davis, Scott C.; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

    2008-02-01

    Aminolevulinic Acid (ALA) is a prodrug which can be administered to cells, animals or patients after which it is transformed via the Heme synthesis pathway into the fluorescent molecule Protoporphyrin IX (PpIX). PpIX has been shown to be useful as both a photosensitizer for photodynamic therapy (PDT) and as a fluorescence imaging contrast agent. The ALA-PpIX system not only provides contrast for fluorescence imaging but also gives information about the metabolic activity of the imaged tissue and thus could be useful for monitoring cancer therapy. In the current study skin photobleaching was examined to determine if PpIX fluorescence contrast in malignant brain tumors could be better visualized noninvasively. Red light photobleaching decreased skin PpIX fluorescence and increased the ability to noninvasively quantify PpIX fluorescence in murine gliomas, as in vivo measurements of mean PpIX fluorescence more closely matched ex vivo quantification following skin photobleaching. Three doses of blue light photobleaching (4 J/cm2, 8 J/cm2 and 12 J/cm2) were tested and determined to give similar levels of skin photobleaching as well as a similar window of decreased skin PpIX fluorescence for noninvasive fluorescence imaging following the photobleaching dose administration.

  17. Constellation's First Flight Test: Ares I-X

    NASA Technical Reports Server (NTRS)

    Davis, Stephan R.; Askins, Bruce R.

    2010-01-01

    On October 28, 2009, NASA launched Ares I-X, the first flight test of the Constellation Program that will send human beings to the Moon and beyond. This successful test is the culmination of a three-and-a-half-year, multi-center effort to design, build, and fly the first demonstration vehicle of the Ares I crew launch vehicle, the successor vehicle to the Space Shuttle. The suborbital mission was designed to evaluate the atmospheric flight characteristics of a vehicle dynamically similar to Ares I; perform a first stage separation and evaluate its effects; characterize and control roll torque; stack, fly, and recover a solid-motor first stage testing the Ares I parachutes; characterize ground, flight, and reentry environments; and develop and execute new ground hardware and procedures. Built from existing flight and new simulator hardware, Ares I-X integrated a Shuttle-heritage four-segment solid rocket booster for first stage propulsion, a spacer segment to simulate a five-segment booster, Peacekeeper axial engines for roll control, and Atlas V avionics, as well as simulators for the upper stage, crew module, and launch abort system. The mission leveraged existing logistical and ground support equipment while also developing new ones to accommodate the first in-line rocket for flying astronauts since the Saturn IB last flew from Kennedy Space Center (KSC) in 1975. This paper will describe the development and integration of the various vehicle and ground elements, from conception to stacking in KSC s Vehicle Assembly Building; hardware performance prior to, during, and after the launch; and preliminary lessons and data gathered from the flight. While the Constellation Program is currently under review, Ares I-X has and will continue to provide vital lessons for NASA personnel in taking a vehicle concept from design to flight.

  18. Ares I-X Test Flight Reference Trajectory Development

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Gumbert, Clyde R.; Tartabini, Paul V.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I crew launch vehicle. Ares I is a two stage to orbit launch vehicle that provides crew access to low Earth orbit for NASA's future manned exploration missions. The Ares I first stage consists of a Shuttle solid rocket motor (SRM) modified to include an additional propellant segment and a liquid propellant upper stage with an Apollo J2X engine modified to increase its thrust capability. The modified propulsion systems were not available for the first test flight, thus the test had to be conducted with an existing Shuttle 4 segment reusable solid rocket motor (RSRM) and an inert Upper Stage. The test flight's primary objective was to demonstrate controllability of an Ares I vehicle during first stage boost and the ability to perform a successful separation. In order to demonstrate controllability, the Ares I-X ascent control algorithms had to maintain stable flight throughout a flight environment equivalent to Ares I. The goal of the test flight reference trajectory development was to design a boost trajectory using the existing RSRM that results in a flight environment equivalent to Ares I. A trajectory similarity metric was defined as the integrated difference between the Ares I and Ares I-X Mach versus dynamic pressure relationships. Optimization analyses were performed that minimized the metric by adjusting the inert upper stage weight and the ascent steering profile. The sensitivity of the optimal upper stage weight and steering profile to launch month was also investigated. A response surface approach was used to verify the optimization results. The analyses successfully defined monthly ascent trajectories that matched the Ares I reference trajectory dynamic pressure versus Mach number relationship to within 10% through Mach 3.5. The upper stage weight required to achieve the match was found to be feasible and varied less than 5% throughout the year. The paper will discuss the flight

  19. Effect of IX dosing on polypropylene and PVDF membrane fouling control.

    PubMed

    Myat, Darli Theint; Mergen, Max; Zhao, Oliver; Stewart, Matthew B; Orbell, John D; Merle, Tony; Croué, Jean-Philippe; Gray, Stephen

    2013-07-01

    The performance of ion exchange (IX) resin for organics removal from wastewater was assessed using advanced characterisation techniques for varying doses of IX. Organic characterisation using liquid chromatography with a photodiode array (PDA) and fluorescence spectroscopy (Method A), and UV254, organic carbon and organic nitrogen detectors (Method B), was undertaken on wastewater before and after magnetic IX treatment. Results showed partial removal of the biopolymer fraction at high IX doses. With increasing concentration of IX, evidence for nitrogen-containing compounds such as proteins and amino acids disappeared from the LC-OND chromatogram, complementary to the fluorescence response. A greater fluorescence response of tryptophan-like proteins (278 nm/343 nm) for low IX concentrations was consistent with aggregation of tryptophan-like compounds into larger aggregates, either by self-aggregation or with polysaccharides. Recycling of IX resin through multiple adsorption steps without regeneration maintained the high level of humics removal but there was no continued removal of biopolymer. Subsequent membrane filtration of the IX treated waters resulted in complex fouling trends. Filtration tests with either polypropylene (PP) or polyvinylidene fluoride (PVDF) membranes showed higher rates of initial fouling following treatment with high IX doses (10 mL/L) compared to filtration of untreated water, while treatment with lower IX doses resulted in decreased fouling rates relative to the untreated water. However, at longer filtration times the rate of fouling of IX treated waters was lower than untreated water and the relative fouling rates corresponded to the amount of biopolymer material in the feed. It was proposed that the mode of fouling changed from pore constriction during the initial filtration period to filter cake build up at longer filtration times. The organic composition strongly influenced the rate of fouling during the initial filtration period due to

  20. Carbonic Anhydrase Inhibitors. Part 551 Metal Complexes of 1,3,4-Thiadiazole-2-Sulfonamide Derivatives: In Vitro Inhibition Studies With Carbonic Anhydrase Isozymes I, II and IV

    PubMed Central

    Scozzafava, Andrea; Briganti, Fabrizio; Ilies, Marc A.; Jitianu, Andrei

    1998-01-01

    Coordination compounds of 5-chloroacetamido-1,3,4-thiadiazole-2-sulfonamide (Hcaz) with V(IV), Cr(lll), Fe(ll), Co(ll), Ni(ll) and Cu(ll) have been prepared and characterized by standard procedures (spectroscopic, magnetic, EPR, thermogravimetric and conductimetric measurements). Some of these compounds showed very good in vitro inhibitory properties against three physiologically relevant carbonic anhydrase (CA)isozymes, i.e., CA I, II, and IV. The differences between these isozymes in susceptibility to inhibition by these metal complexes is discussed in relationship to the characteristic features of their active sites, and is rationalized in terms useful for developing isozyme-specific CA inhibitors. PMID:18475829

  1. Characterization of three homologs of the large subunit of the magnesium chelatase from Chlorobaculum tepidum and interaction with the magnesium protoporphyrin IX methyltransferase.

    PubMed

    Johnson, Ethan T; Schmidt-Dannert, Claudia

    2008-10-10

    Green bacteria synthesize several types of (bacterio)chlorophylls for the assembly of functional photosynthetic reaction centers and antenna complexes. A distinctive feature of green bacteria compared with other photosynthetic microbes is that their genomes contain multiple homologs of the large subunit (BchH) of the magnesium chelatase which is a three-subunit enzyme complex (BchH, BchD, and BchI) that inserts magnesium into protoporphyrin IX as the first committed step of (bacterio)chlorophyll biosynthesis. There is speculation that the additional BchH homologs may regulate the biosynthesis of each type of chlorophyll, although the biochemical properties of the different magnesium chelatase complexes from a single species of green bacteria have not yet been compared. In this study, we investigated the activities of all three chelatase complexes from the green sulfur bacterium Chlorobaculum tepidum and interactions with the next enzyme in the pathway, magnesium protoporphyrin IX methyltransferase (BchM). Although all three chelatase complexes insert magnesium into protoporphyrin IX, the activities range by a factor of 10(5). Further, there are differences in the interactions between the BchH homologs and BchM; two of the subunits increase the methyltransferase activity by 30-60%, and the third decreases it by 30%. Expression of the chelatase complexes alone and together with BchM in Escherichia coli overproducing protoporphyrin IX suggests that the chelatase is the rate-limiting enzyme. We observed that BchM uses protoporphyrin IX without bound metal as a substrate. Our results conflict with expectations generated by previous gene inactivation studies and suggest a complex regulation of chlorophyll biosynthesis in green bacteria.

  2. Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome

    SciTech Connect

    Doyen, J.; Trastour, C.; Ettore, F.; Peyrottes, I.; Toussant, N.; Gal, J.; Ilc, K.; Roux, D.; Parks, S.K.; Ferrero, J.M.; Pouysségur, J.

    2014-08-15

    Highlights: • Glycolytic markers are highly expressed in triple negative breast cancers. • Lactate/H{sup +} symporter MCT4 demonstrated the strongest deleterious impact on survival. • MCT4 should serve as a new prognostic factor in node-negative breast cancers. - Abstract: Background: {sup 18}Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer. Methods: Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohorts of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively. Results: In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR = 0.47, P = 0.02) and overall-survival (HR = 0.38, P = 0.002). These results were confirmed in the independent cohort of 127 cancer patients. Conclusion: Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H{sup +} symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.

  3. Eriocitrin and Apigenin as New Carbonic Anhydrase VA Inhibitors from a Virtual Screening of Calabrian Natural Products.

    PubMed

    Gidaro, Maria Concetta; Alcaro, Francesca; Carradori, Simone; Costa, Giosuè; Vullo, Daniela; Supuran, Claudiu T; Alcaro, Stefano

    2015-04-01

    In this work, we performed a structure-based virtual screening against five carbonic anhydrase isoforms using, as a ligand library, natural components of Citrus bergamia (Bergamot) and Allium cepa var. Tropea (red onion) sources, which are some typical Calabrian products. The most relevant Bergamot and red onion components, identified as potentially new hits by means of the computational work, were submitted to in vitro tests in order to confirm the ability to exert the predicted biological activity. Apigenin and eriocitrin were identified as new potent inhibitors of human carbonic anhydrase VA isozyme.

  4. Dissolved carbonic anhydrase for enhancing post-combustion carbon dioxide hydration in aqueous ammonia

    SciTech Connect

    Collett, James R.; Heck, Robert W.; Zwoster, Andy

    2011-04-01

    Aqueous ammonia solvents that capture CO2 as ionic complexes of carbonates with ammonium have recently been advanced as alternatives to amine-based solvents due to their lower energy requirements for thermal regeneration. In ammonia based solvents, the hydration of CO2 to form bicarbonate may become a rate-limiting step as the CO2 loading increases and the resulting pH level of the solvent decreases. Variants of the enzyme carbonic anhydrase can accelerate the reversible hydration of CO2 to yield bicarbonate by more than 10(6)-fold. The possible benefit of bovine carbonic anhydrase (BCA) addition to solutions of aqueous ammonia to enhance CO2 hydration was investigated in semi-batch reactions within continuously stirred tank reactors or in a bubble column gas-liquid contactor. Adding 154 mg/liter of BCA to 2 M aqueous ammonia provided a 34.1% overall increase in the rate of CO2 hydration (as indicated by the production of [H+]) as the pH declined from 9.6 to 8.6 during sparging with a 15% CO2, 85% N-2 gas at a flow rate of 3 lpm. The benefits of adding BCA to enhance CO2 hydration were only discernable below similar to pH 9. The implications of the apparent pH limitations on the utility of BCA are discussed in the context of absorber unit operation design. Possible embodiments of carbonic anhydrase as either an immobilized catalyst or as a dissolved, recirculating catalyst in potential plant scale aqueous ammonia systems are considered as well. (C) 2010 Published by Elsevier Ltd.

  5. Ares I-X Separation and Reentry Trajectory Analyses

    NASA Technical Reports Server (NTRS)

    Tartabini, Paul V.; Starr, Brett R.

    2011-01-01

    The Ares I-X Flight Test Vehicle was launched on October 28, 2009 and was the first and only test flight of NASA s two-stage Ares I launch vehicle design. The launch was successful and the flight test met all of its primary and secondary objectives. This paper discusses the stage separation and reentry trajectory analysis that was performed in support of the Ares I-X test flight. Pre-flight analyses were conducted to assess the risk of stage recontact during separation, to evaluate the first stage flight dynamics during reentry, and to define the range safety impact ellipses of both stages. The results of these pre-flight analyses were compared with available flight data. On-board video taken during flight showed that the flight test vehicle successfully separated without any recontact. Reconstructed trajectory data also showed that first stage flight dynamics were well characterized by pre-flight Monte Carlo results. In addition, comparisons with flight data indicated that the complex interference aerodynamic models employed in the reentry simulation were effective in capturing the flight dynamics during separation. Finally, the splash-down locations of both stages were well within predicted impact ellipses.

  6. Ares I-X Upper Stage Simulator Residual Stress Analysis

    NASA Technical Reports Server (NTRS)

    Raju, Ivatury S.; Brust, Frederick W.; Phillips, Dawn R.; Cheston, Derrick

    2008-01-01

    The structural analyses described in the present report were performed in support of the NASA Engineering and Safety Center (NESC) Critical Initial Flaw Size (CIFS) assessment for the Ares I-X Upper Stage Simulator (USS) common shell segment. An independent assessment was conducted to determine the critical initial flaw size (CIFS) for the flange-to-skin weld in the Ares I-X Upper Stage Simulator (USS). The Ares system of space launch vehicles is the US National Aeronautics and Space Administration s plan for replacement of the aging space shuttle. The new Ares space launch system is somewhat of a combination of the space shuttle system and the Saturn launch vehicles used prior to the shuttle. Here, a series of weld analyses are performed to determine the residual stresses in a critical region of the USS. Weld residual stresses both increase constraint and mean stress thereby having an important effect on fatigue and fracture life. The results of this effort served as one of the critical load inputs required to perform a CIFS assessment of the same segment.

  7. Atomic Data and Spectral Line Intensities for Ca IX

    NASA Technical Reports Server (NTRS)

    Landi, E.; Bhatia, A. K.

    2012-01-01

    Electron impact collision strengths, energy levels, oscillator strengths and spontaneous radiative decay rates are calculated for Ca IX. We include in the calculations the 33 lowest configurations in the n = 3, 4, 5 complexes, corresponding to 283 fine structure levels in the 3l3l ', 3l4l'' and 3l4l''' configurations, where l,l' = s, p, d, l '' = s, p, d, f and l''' = s, p, d, f, g. Collision strengths are calculated at five incident energies for all transitions: 5.8, 13.6, 24.2, 38.6 and 57.9 Ry above the threshold of each transition. An additional energy, very close to the transition threshold, has been added, whose value is between 0.0055 Ry and 0.23 Ry depending on the levels involved. Calculations have been carried out using the Flexible Atomic Code and the distorted wave approximation. Excitation rate coefficients are calculated as a function of electron temperature by assuming a Maxwellian electron velocity distribution. Using the excitation rate coefficients and the radiative transition rates calculated in the present work, statistical equilibrium equations for level populations are solved at electron densities covering the 10(exp 8)-10(exp 14)/cubic cm range and at an electron temperature of log T(sub e)(K)=5.8, corresponding to the maximum abundance of Ca IX. Spectral line intensities are calculated, and their diagnostic relevance is discussed.

  8. 5-ALA/PpIX fluorescence detection of gastrointestinal neoplasia

    NASA Astrophysics Data System (ADS)

    Borisova, Ekaterina G.; Vladimirov, Borislav; Terziev, Ivan; Ivanova, Radina; Avramov, Latchezar

    2009-07-01

    In the recent study delta-ALA/PpIX is used as fluorescent marker for dysplasia and tumor detection in esophagus, stomach and colon. ALA is administered per os six to eight (depending on the lesion location) hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for the LED to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to re-absorption of oxy-hemoglobin in this spectral area. Endogenous and exogenous fluorescence spectra are used to develop simple but effective algorithm, based on dimensionless ratio of the signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues. Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

  9. A Case of Carbonic Anhydrase Type 2 Deficiency Syndrome with Autistic Disorder

    PubMed Central

    KILIÇ, Birim Günay; UĞUR, Çağatay; SADAY DUMAN, Nagihan; AKÇAKIN, Melda

    2014-01-01

    Carbonic Anhydrase Type II Deficiency Syndrome (CADS) is a disease with an autosomal recessive inheritance that mainly includes characteristics of osteopetrosis, renal tubular acidosis and cerebral calcification. Pathological fractures, poor vision due to cranial nerve pressure, wide forehead, disproportionate mouth and jaw, physical and mental developmental delay are other features. In this paper, we present the case of a patient who was referred to our department with a diagnosis of CADS and diagnosed with autistic disorder after a psychiatric evaluation. We performed a detailed literature search, however, we did not find any report of co-existence of CADS (osteopetrosis intermediate type) and autistic disorder.

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

    PubMed Central

    Lionetto, Maria Giulia; Caricato, Roberto; Giordano, Maria Elena; Erroi, Elisa; Schettino, Trifone

    2012-01-01

    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. PMID:23202827

  11. Carbonic anhydrase II-induced selection of inhibitors from a dynamic combinatorial library of Schiff's bases.

    PubMed

    Nasr, Gihane; Petit, Eddy; Supuran, Claudiu T; Winum, Jean-Yves; Barboiu, Mihail

    2009-11-01

    A dynamic combinatorial library (DCL) has been generated under thermodynamic control by using the aminocarbonyl/imine interconversion as reversible chemistry, combined with non-covalent binding within the active site of the metalloenzyme human carbonic anhydrase II (hCA II, EC 4.2.1.1). The high affinity of hCA II isozyme towards some sulfonamide inhibitors obtained here was used to select from the dynamic library specific inhibitors of this isoform. These results point out to the possibility of identifying sulfonamide amplified compounds presenting potent inhibition and high yield of formation in the presence of the isoform(s) towards which the inhibitors were designed.

  12. A Place on the Team: The Triumph and Tragedy of Title IX

    ERIC Educational Resources Information Center

    Suggs, Welch

    2006-01-01

    "A Place on the Team" is the inside story of how Title IX revolutionized American sports. The federal law guaranteeing women's rights in education, Title IX opened gymnasiums and playing fields to millions of young women previously locked out. Journalist Welch Suggs chronicles both the law's successes and failures-the exciting…

  13. "What Do I Think about Title IX?" Voices from a University Community

    ERIC Educational Resources Information Center

    Paule-Koba, Amanda L.; Harris, Othello; Freysinger, Valeria J.

    2013-01-01

    Despite the apparent benefits of Title IX, the implementation of the law remains controversial, and there are divergent beliefs regarding its impact on collegiate sport. The purpose of this study was to examine how members of a university community, whose intercollegiate sport programs have changed, perceive and make sense of Title IX and the…

  14. Critical Issue Bibliography (CRIB) Sheet: Title IX of the Educational Amendments of 1972.

    ERIC Educational Resources Information Center

    ERIC Clearinghouse on Higher Education, Washington, DC.

    This CRitical Issue Bibliography (CRIB) Sheet cites resources that give an overview of Title IX legislation and offer information about compliance, litigation, and related issues. Title IX of the Education Amendments of 1972 protects individuals from gender-based discrimination in education programs or activities that receive federal financial…

  15. Not Second-Class: Title IX, Equity, and Girls' High School Sports

    ERIC Educational Resources Information Center

    Stader, David L.; Surface, Jeanne L.

    2014-01-01

    Title IX is designed to protect students from discrimination based on sex in any educational institution that receives financial assistance. This article focuses on Title IX as it applies to high school athletic programs by considering the trial of a high school district in California. A federal court found considerable inequalities between boys…

  16. Title IX: What It Means and Doesn't Mean to Athletic Programs.

    ERIC Educational Resources Information Center

    Dunkle, Margaret C.

    This document examines equal opportunity for women in athletic programs--one of the most closely scrutinized and least understood issues of Title IX. Areas considered are: (1) basic provisions of Title IX; (2) the self-evaluation requirement; (3) grievance procedures; (4) the "adjustment period"; (5) overall equal athletic opportunity;…

  17. The Unenforced Law: Title IX Activity by Federal Agencies other than HEW. Report.

    ERIC Educational Resources Information Center

    Balles, Nancy J.

    The purposes of this report are to identify which federal agencies other than the Department of Health, Education, and Welfare have educational programs covered by Title IX; to survey those agencies to determine if they are enforcing Title IX by means of regulations, orders, or termination of funding; to identify problems of intragovernmental…

  18. Derivation of a triple mosaic adenovirus based on modification of the minor capsid protein IX

    SciTech Connect

    Tang Yizhe; Le, Long P.; Matthews, Qiana L.; Han Tie; Wu Hongju; Curiel, David T.

    2008-08-01

    Adenoviral capsid protein IX (pIX) has been shown to be a potential locale to insert targeting, imaging-related and therapeutic modalities by genetic modification. Recent evidences suggested that capsid protein mosaicism could be a promising strategy for improving the utility of Ad vector. In this study, we explored a method to genetically generate triple pIX mosaic Ad serotype 5 (Ad5) displaying three types of pIX on a single virion. pIXs were modified at their carboxy termini with a Flag sequence, a hexahistidine sequence (His{sub 6}) or a monomeric red fluorescent protein (mRFP1), respectively. Western blotting analysis and fluorescence microscopy of the purified recombinant viruses indicated that all three modified pIXs were incorporated into the viral particles. Immuno-gold electron microscopy (EM) further confirmed that three types of pIX indeed co-existed on an individual virion. These results firstly validated a triple mosaic capsid configuration on pIX, and demonstrated the possibility of further radical design.

  19. Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Olson, Jonathan D.; Kanick, Stephen C.; Bravo, Jaime J.; Roberts, David W.; Paulsen, Keith D.

    2016-03-01

    Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

  20. Almost As Fairly: The First Year of Title IX Implementation in Six Southern States. A Report.

    ERIC Educational Resources Information Center

    American Friends Service Committee, Columbia, SC. Southeastern Public Education Program.

    Volunteers from community organizations in six southern states monitored 21 school districts to find their districts' initial answer to Title IX, federal legislation barring sex discrimination. The actual monitoring of the 21 districts was completed in the late spring of 1976, with data covering the first year of Title IX implementation. The…

  1. 40 CFR Appendix Ix to Part 266 - Methods Manual for Compliance With the BIF Regulations

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Methods Manual for Compliance With the BIF Regulations IX Appendix IX to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) STANDARDS FOR THE MANAGEMENT OF SPECIFIC HAZARDOUS WASTES AND SPECIFIC TYPES OF HAZARDOUS WASTE...

  2. Modelling topical photodynamic therapy treatment including the continuous production of Protoporphyrin IX

    NASA Astrophysics Data System (ADS)

    Campbell, C. L.; Brown, C. T. A.; Wood, K.; Moseley, H.

    2016-11-01

    Most existing theoretical models of photodynamic therapy (PDT) assume a uniform initial distribution of the photosensitive molecule, Protoporphyrin IX (PpIX). This is an adequate assumption when the prodrug is systematically administered; however for topical PDT this is no longer a valid assumption. Topical application and subsequent diffusion of the prodrug results in an inhomogeneous distribution of PpIX, especially after short incubation times, prior to light illumination. In this work a theoretical simulation of PDT where the PpIX distribution depends on the incubation time and the treatment modality is described. Three steps of the PpIX production are considered. The first is the distribution of the topically applied prodrug, the second in the conversion from the prodrug to PpIX and the third is the light distribution which affects the PpIX distribution through photobleaching. The light distribution is modelled using a Monte Carlo radiation transfer model and indicates treatment depths of around 2 mm during daylight PDT and approximately 3 mm during conventional PDT. The results suggest that treatment depths are not only limited by the light penetration but also by the PpIX distribution.

  3. Perceptions of Women's Teams Coaches Regarding Gender Equity and Title IX Compliance in Community Colleges

    ERIC Educational Resources Information Center

    Kenney, Cynthia A.

    2013-01-01

    Title IX was enacted over 40 years ago, and although there have been marked increases in the number of girls and women participating in athletics at every level, gender equity in athletics continues to be a concern. This is especially evident at the community college level. Title IX requires equity in the areas of opportunities for participation,…

  4. Title IX Compliance at Two-Year Colleges: An Analysis of Perceived Barriers and Strategies

    ERIC Educational Resources Information Center

    Causby, Cory Scott

    2010-01-01

    Although Title IX legislation has been in effect since 1972 and has created unprecedented positive change on intercollegiate athletics, educational institutions have still had difficulty meeting the basic requirements set forth by Title IX and ensuring gender equity in their athletic programs. Additionally, specific research has been largely…

  5. "What do I think about Title IX?" Voices from a university community.

    PubMed

    Paule-Koba, Amanda L; Harris, Othello; Freysinger, Valeria J

    2013-03-01

    Despite the apparent benefits of Title IX, the implementation of the law remains controversial, and there are divergent beliefs regarding its impact on collegiate sport. The purpose of this study was to examine how members of a university community, whose intercollegiate sport programs have changed, perceive and make sense of Title IX and the changes it incurred. Using a gendered lens (Birrell, 2000) and qualitative interviews with 13 individuals connected to the university community, insight was gained into their perceptions of Title IX and women's and men's athletic opportunities. The theme of opportunity gained and lost was central to how these individuals perceive Title IX and resonates with the liberal (feminist) concerns out of which Title IX was born.

  6. Evidence for a prevalent dimorphism in the activation peptide of human coagulation factor IX.

    PubMed Central

    McGraw, R A; Davis, L M; Noyes, C M; Lundblad, R L; Roberts, H R; Graham, J B; Stafford, D W

    1985-01-01

    We have independently isolated and characterized cDNA and genomic clones for the human coagulation factor IX. Sequence analysis in both cases indicates that threonine is encoded by the triplet ACT as the third residue of the activation peptide. This is in agreement with some earlier reports but in disagreement with others that show the alanine triplet GCT at this position. The discrepancy can thus be accounted for by natural variation of a single nucleotide in the normal population. Amino acid sequence analyses of activated factor IX from plasma samples of four individuals yielded two cases of alanine and two cases of threonine at the third position of the activation peptide. In factor IX from pooled plasma and in factor IX from a heterozygous individual, however, both alanine and threonine were found. Taken together, the findings show that a prevalent nondeleterious dimorphism exists in the activation peptide of human coagulation factor IX. PMID:3857619

  7. Ares I-X Flight Test Vehicle: Stack 5 Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Danel R.

    2010-01-01

    Ares I-X was the first flight test vehicle used in the development of NASA's Ares I crew launch vehicle. The Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Three modal tests were defined to verify the dynamic finite element model of the Ares I-X flight test vehicle. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report focuses on the first modal test that was performed on the top section of the vehicle referred to as Stack 5, which consisted of the spacecraft adapter, service module, crew module and launch abort system simulators. This report describes the test requirements, constraints, pre-test analysis, test operations and data analysis for the Ares I-X Stack 5 modal test.

  8. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  9. Ares I-X Flight Test Vehicle:Stack 1 Modal Test

    NASA Technical Reports Server (NTRS)

    Buehrle, Ralph D.; Templeton, Justin D.; Reaves, Mercedes C.; Horta, Lucas G.; Gaspar, James L.; Bartolotta, Paul A.; Parks, Russel A.; Lazor, Daniel R.

    2010-01-01

    Ares I-X was the first flight test vehicle used in the development of NASA s Ares I crew launch vehicle. The Ares I-X used a 4-segment reusable solid rocket booster from the Space Shuttle heritage with mass simulators for the 5th segment, upper stage, crew module and launch abort system. Three modal tests were defined to verify the dynamic finite element model of the Ares I-X flight test vehicle. Test configurations included two partial stacks and the full Ares I-X flight test vehicle on the Mobile Launcher Platform. This report focuses on the second modal test that was performed on the middle section of the vehicle referred to as Stack 1, which consisted of the subassembly from the 5th segment simulator through the interstage. This report describes the test requirements, constraints, pre-test analysis, test operations and data analysis for the Ares I-X Stack 1 modal test.

  10. Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

    PubMed Central

    Laitala, T; Väänänen, H K

    1994-01-01

    The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

  11. Dual-wavelength excitation for fluorescence-based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood.

    PubMed

    Hennig, Georg; Gruber, Christian; Vogeser, Michael; Stepp, Herbert; Dittmar, Stephan; Sroka, Ronald; Brittenham, Gary M

    2014-07-01

    Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron-restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual-wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual-wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (rs = 0.943, p < 0.0001; range 37-689 μmol ZnPP/mol heme, 84-1238 nmol/L) and for PPIX (rs = 0.959, p < 0.0001; range 42-4212 μmol PPIX/mol heme, 93-5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single-wavelength excitation and with commercial front-face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual-wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX.

  12. 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence in Meningioma: Qualitative and Quantitative Measurements In Vivo

    PubMed Central

    Valdes, Pablo A.; Bekelis, Kimon; Harris, Brent T.; Wilson, Brian C.; Leblond, Frederic; Kim, Anthony; Simmons, Nathan E.; Erkmen, Kadir; Paulsen, Keith D.; Roberts, David W.

    2014-01-01

    BACKGROUND The use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence has shown promise as a surgical adjunct for maximizing the extent of surgical resection in gliomas. To date, the clinical utility of 5-ALA in meningiomas is not fully understood, with most descriptive studies using qualitative approaches to 5-ALA-PpIX. OBJECTIVE To assess the diagnostic performance of 5-ALA-PpIX fluorescence during surgical resection of meningioma. METHODS ALA was administered to 15 patients with meningioma undergoing PpIX fluorescence-guided surgery at our institution. At various points during the procedure, the surgeon performed qualitative, visual assessments of fluorescence by using the surgical microscope, followed by a quantitative fluorescence measurement by using an intra-operative probe. Specimens were collected at each point for subsequent neuropathological analysis. Clustered data analysis of variance was used to ascertain a difference between groups, and receiver operating characteristic analyses were performed to assess diagnostic capabilities. RESULTS Red-pink fluorescence was observed in 80% (12/15) of patients, with visible fluorescence generally demonstrating a strong, homogenous character. Quantitative fluorescence measured diagnostically significant PpIX concentrations (CPpIx) in both visibly and nonvisibly fluorescent tissues, with significantly higher CPpIx in both visibly fluorescent (P < .001) and tumor tissue (P = .002). Receiver operating characteristic analyses also showed diagnostic accuracies up to 90% for differentiating tumor from normal dura. CONCLUSION ALA-induced PpIX fluorescence guidance is a potential and promising adjunct in accurately detecting neoplastic tissue during meningioma resective surgery. These results suggest a broader reach for PpIX as a biomarker for meningiomas than was previously noted in the literature. PMID:23887194

  13. Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae.

    PubMed

    Zito, Francesca; Koop, Demian; Byrne, Maria; Matranga, Valeria

    2015-09-01

    Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non-skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC-specific marker. Strikingly, the AZ-treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.

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

    PubMed

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Carbonic Anhydrase is Required for Statoconia Homeostasis in Organ Cultures of Statocysts from Aplysia californica

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

    A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (LI5) medium supplemented with salts and Aplysia haemolymph for four days at 17 C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(exp -4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially, via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.

  17. CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

    PubMed

    Semenihin, A V; Zolotareva, O K

    2015-01-01

    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.

  18. The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II.

    PubMed

    Sterling, Deborah; Brown, Nathan J D; Supuran, Claudiu T; Casey, Joseph R

    2002-11-01

    COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 +/- 1 mM H+ equivalents/min was inhibited 53 +/- 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 +/- 4% without effect on DRA transport activity (105 +/- 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.

  19. Maleate-induced bicarbonaturia in the dog: a carbonic anhydrase-independene effect.

    PubMed

    Gougoux, A; Lemieux, G; Lavoie, N

    1976-10-01

    Studies were performed to characterize the renal effects of maleate in anesthetized dogs. Following the intravenous administration of maleate or maleic acid (50 mg/kg), mean fractional bicarbonate excretion (CHCO3/GFR) rose to as high as 26%. Na, K, and phosphate excretion also increased markedly, whereas C1 excretion remained low. An initial transient fall in urinary pH from 6.53 to 6.13 contrasted sharply with the rapid alkalinization of the urine induced by acetazolamide administration. During saline expansion CHCO3/GFR rose from 4 to 37% after maleate administration, whereas Cl excretion did not change significantly. During continuous carbonic anhydrase inhibition with acetazolamide, maleate administration resulted in a further rise in CHCO3/GFR from 22 to 35%. Whereas CPO4/GFR increased only from 1 to 3% during acetazolamide administration, this ratio reached 75% following the addition of maleate. Fumarate, the transisomer of maleate, and malonate, a well-known inhibitor of Krebs cycle, failed to affect bicarbonate excretion. This study demonstrates that maleate inhibits the fraction of bicarbonate reabsorption uncatalyzed by carbonic anhydrase. Impaired anionic reabsorption of bicarbonate or accelerated passive backflux of this ion into proximal tubular lumen are the two mechanisms that best explain the bicarbonaturia induced by maleate.

  20. A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase.

    PubMed

    Iqbal, Shoaib; Nisar-ur-Rahman; Iqbal, Jamshed

    2014-01-01

    In the current study, capillary electrophoresis (CE)-based enzyme assay for characterization and inhibition study of bovine carbonic anhydrase II (bCA II) was developed. The developed method is the first CE assay for carbonic anhydrase (CA). The method was optimized in order to get short analysis time, minimal sample volume consumption, and high resolution of substrate and product. The CE conditions were optimized as follows: fused-silica capillary (30 cm effective length×75 μm i.d.), pressure injection for 5s, 20mM sodium borate buffer (pH 9.0), constant voltage of 15 kV, constant capillary temperature of 25 °C, and detection at 260 nm. For precise measurements, uridine was used as an internal standard during optimization of the CE methods. The limits of detection and quantification for p-nitrophenyl acetate (p-NPA) were 3.01 and 9.12 μM, respectively, whereas for p-nitrophenolate they were 2.05 and 6.22 μM, respectively. The performance of the developed method was confirmed by determination of kinetic parameters (i.e., K(m) and V(max) of bCA for p-NPA); the inhibition constant (K(i)) was determined for furosemide, a standard inhibitor of CA. The new method proved to be fast and efficient, and it can be used for the investigation of inhibitors of all isoforms of CAs.

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

    PubMed Central

    Francis, Sheena A. M.; Taylor-Wells, Jennina; Gross, Aaron D.; Bloomquist, Jeffrey R.

    2016-01-01

    The physiological role of carbonic anhydrases in pH and ion regulation is crucial to insect survival. We examined the toxic and neurophysiological effects of five carbonic anhydrase inhibitors (CAIs) against Aedes aegypti. The 24 h larvicidal toxicities followed this rank order of potency: dichlorphenamide > methazolamide > acetazolamide = brinzolamide = dorzolamide. Larvicidal activity increased modestly in longer exposures, and affected larvae showed attenuated responses to probing without overt tremors, hyperexcitation, or convulsions. Acetazolamide and dichlorphenamide were toxic to adults when applied topically, but were of low potency and had an incomplete effect (<50% at 300 ng/mosquito) even after injection. Dichlorphenamide was also the most toxic compound when fed to adult mosquitoes, and they displayed loss of posture and occasionally prolonged fluttering of the wings. Co-exposure with 500 ng of the synergist piperonyl butoxide (PBO) increased the toxicity of dichlorphenamide ca. two-fold in feeding assays, indicating that low toxicity was not related to oxidative metabolism. Dichlorphenamide showed mild depolarizing and nerve discharge actions on insect neuromuscular and central nervous systems, respectively. These effects were increased in low buffer salines, indicating they were apparently related to loss of pH control in these tissues. Overall, sulfonamides displayed weak insecticidal properties on Aedes aegypti and are weak lead compounds. PMID:28025488

  2. Carbonic anhydrase, a respiratory enzyme in the gills of the shore crab Carcinus maenas

    NASA Astrophysics Data System (ADS)

    Böttcher, K.; Siebers, D.; Sender, S.

    1995-03-01

    This paper summarizes investigations on the enzyme carbonic anhydrase (CA) in the gills of the osmoregulating shore crab Carcinus maenas. Carbonic anhydrase, an enzyme catalyzing the reversible hydration of CO2 to HCO3 - and H+, is localized with highest activities in the posterior salt-transporting gills of the shore crab- and here CA activity is strongly dependent on salinity. Contrary to the earlier hypothesis established for the blue crab Callinectes sapidus that cytoplasmic branchial CA provides the counter ions HCO3 - and H+ for apical exchange against Na+ and Cl-, the involvement of CA in NaCl uptake mechanisms can be excluded in Carcinus. Differential and density gradient centrifugations indicate that branchial CA is a predominantly membrane-associated protein. Branchial CA was greatly inhibited by the sulfonamide acetazolamide (AZ) Ki=2.4·10-8 mol/l). Using the preparation of the isolated perfused gill, application of 10-4 mol/l AZ resulted in an 80% decrease of CO2/HCO3 - excretion. Thus we conclude that CA is localized in plasma membranes, maintaining the CO2 gradient by accelerating adjustment of the pH-dependent CO2/HCO3 - equilibrium.

  3. Effects of dopaminergic compounds on carbonic anhydrase isozymes I, II, and VI.

    PubMed

    Sentürk, Murat; Ekinci, Deniz; Göksu, Süleyman; Supuran, Claudiu T

    2012-06-01

    Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K(i) values of the molecules 3-6 were in the range of 41.12-363 μM against hCA I, of 0.381-470 μM against hCA II and of 0.578-1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K(A) values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.

  4. Two developmental switches in GABAergic signalling: the K+–Cl− cotransporter KCC2 and carbonic anhydrase CAVII

    PubMed Central

    Rivera, Claudio; Voipio, Juha; Kaila, Kai

    2005-01-01

    GABAergic signalling has the unique property of ‘ionic plasticity’, which is based on short-term and long-term changes in the Cl− and HCO3− ion concentrations in the postsynaptic neurones. While short-term ionic plasticity is caused by activity-dependent, channel-mediated anion shifts, long-term ionic plasticity depends on changes in the expression patterns and kinetic regulation of molecules involved in anion homeostasis. During development the efficacy and also the qualitative nature (depolarization/excitation versus hyperpolarization/inhibition) of GABAergic transmission is influenced by the neuronal expression of two key molecules: the chloride-extruding K+–Cl− cotransporter KCC2, and the cytosolic carbonic anhydrase (CA) isoform CAVII. In rat hippocampal pyramidal neurones, a steep up-regulation of KCC2 accounts for the ‘developmental switch’, which converts depolarizing and excitatory GABA responses of immature neurones to classical hyperpolarizing inhibition by the end of the second postnatal week. The immature hippocampus generates large-scale network activity, which is abolished in parallel by the up-regulation of KCC2 and the consequent increase in the efficacy of neuronal Cl− extrusion. At around postnatal day 12 (P12), an abrupt, steep increase in intrapyramidal CAVII expression takes place, promoting excitatory responses evoked by intense GABAergic activity. This is largely caused by a GABAergic potassium transient resulting in spatially widespread neuronal depolarization and synchronous spike discharges. These facts point to CAVII as a putative target of CA inhibitors that are used as antiepileptic drugs. KCC2 expression in adult rat neurones is down-regulated following epileptiform activity and/or neuronal damage by BDNF/TrkB signalling. The lifetime of membrane-associated KCC2 is very short, in the range of tens of minutes, which makes KCC2 ideally suited for mediating GABAergic ionic plasticity. In addition, factors influencing

  5. Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide.

    PubMed

    da Costa Ores, Joana; Sala, Luisa; Cerveira, Guido Picaluga; Kalil, Susana Juliano

    2012-06-01

    This work presents a study of industrially applicable techniques to obtain a biologically supported carbon dioxide capture system, based on the extraction of carbonic anhydrase from bovine blood. Carbonic anhydrase is a metalloenzyme which catalyzes the reversible hydration of carbon dioxide. The objective of this study was to establish conditions to obtain carbonic anhydrase from bovine erythrocytes and apply it in the capture of carbon dioxide. To achieve this, two different purification techniques were evaluated: one by extraction with the organic solvents chloroform and ethanol, where different solvent proportions were studied; and the other by ammonium sulfate precipitation, testing percent saturations between 10% and 80%. Carbon dioxide was enzymatically captured by its precipitation as calcium carbonate with the enzyme obtained by both techniques. The enzyme extracted by ethanol and chloroform showed an activity of 2623 U mL(-1), recovery of 98% and purification factor of 104-fold. That precipitated by ammonium sulfate showed an activity of 2162 U mL(-1), recovery of 66% and purification factor of 1.4-fold using 60% ammonium sulfate saturation. The results obtained in the carbon dioxide capture experiments showed that the carbonic anhydrase extracted in this study not only enhanced the hydration of CO(2), but also promoted the formation of CaCO(3).

  6. Photodynamic action of protoporphyrin IX derivatives on Trichophyton rubrum*

    PubMed Central

    Ramos, Rogério Rodrigo; Kozusny-Andreani, Dora Inês; Fernandes, Adjaci Uchôa; Baptista, Mauricio da Silva

    2016-01-01

    BACKGROUND Dermatophytes are filamentous keratinophilic fungi. Trichophyton rubrum is a prevalent infectious agent in tineas and other skin diseases. Drug therapy is considered to be limited in the treatment of such infections, mainly due to low accessibility of the drug to the tissue attacked and development of antifungal resistance in these microorganisms. In this context, Photodynamic Therapy is presented as an alternative. OBJECTIVE Evaluate, in vitro, the photodynamic activity of four derivatives of Protoporphyrin IX by irradiation with LED 400 nm in T. rubrum. METHOD Assays were subjected to irradiation by twelve cycles of ten minutes at five minute intervals. RESULT Photodynamic action appeared as effective with total elimination of UFCs from the second irradiation cycle. CONCLUSION Studies show that the photodynamic activity on Trichophyton rubrum relates to a suitable embodiment of the photosensitizer, which can be maximized by functionalization of peripheral groups of the porphyrinic ring. PMID:27192510

  7. Protoporphyrin IX: the Good, the Bad, and the Ugly

    PubMed Central

    Sachar, Madhav; Anderson, Karl E.

    2016-01-01

    Protoporphyrin IX (PPIX) is ubiquitously present in all living cells in small amounts as a precursor of heme. PPIX has some biologic functions of its own, and PPIX-based strategies have been used for cancer diagnosis and treatment (the good). PPIX serves as the substrate for ferrochelatase, the final enzyme in heme biosynthesis, and its homeostasis is tightly regulated during heme synthesis. Accumulation of PPIX in human porphyrias can cause skin photosensitivity, biliary stones, hepatobiliary damage, and even liver failure (the bad and the ugly). In this work, we review the mechanisms that are associated with the broad aspects of PPIX. Because PPIX is a hydrophobic molecule, its disposition is by hepatic rather than renal excretion. Large amounts of PPIX are toxic to the liver and can cause cholestatic liver injury. Application of PPIX in cancer diagnosis and treatment is based on its photodynamic effects. PMID:26588930

  8. An Up-Close Look at Ares I-X

    NASA Technical Reports Server (NTRS)

    Leahy, Bart

    2010-01-01

    On October 28, 2009, one day later than the originally planned launch date, the Ares I-X suborbital test flight roared into the Florida sky. Flying its preplanned parabolic arc over the Atlantic, the development test vehicle for the Ares I crew launch vehicle performed as advertised, executing a perfect liftoff, 90-degree roll maneuver, ascent, and separation before its upper and lower stages descended into the ocean 150 miles downrange. This test flight, while carrying no astronauts, marked a major milestone for NASA, which had not flown a test launch of a human-rated rocket since the first flight of the Space Shuttle in 1981. During the flight, over 700 sensors collected over 900 measurements, which NASA will apply to validating the engineering models they used to design the vehicle in the first place. That data, telemetered to the ground and stored in a flight recorder onboard, was the primary "payload" of the mission.

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

    PubMed

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

    2010-04-26

    Topical hypotensive therapy with both timolol and carbonic anhydrase inhibitors has been shown to be efficacious at reducing intraocular pressure. Many prospective studies have also suggested that carbonic anhydrase inhibitors augment ocular blood flow and vascular regulation independent of their hypotensive effects. Although consistent in their findings, these studies must be cautiously interpreted due to the limitations of study design and specific blood flow imaging modalities. The purpose of this review is to appraise and critically evaluate the current body of literature investigating the effects of combined treatment with topical carbonic anhydrase inhibitors and timolol in patients with glaucoma with respect to ocular blood flow, visual function, and optic nerve head structure.

  10. MYStIX: Dynamical evolution of young clusters

    NASA Astrophysics Data System (ADS)

    Kuhn, Michael A.

    2014-08-01

    The spatial structure of young stellar clusters in Galactic star-forming regions provides insight into these clusters’ dynamical evolution---a topic with implications for open questions in star-formation and cluster survival. The Massive Young Star-Forming Complex Study in Infrared and X-ray (MYStIX) provides a sample of >30,000 young stars in star-forming regions (d<3.6 kpc) that contain at least one O-type star. We use the finite mixture model analysis to identify subclusters of stars and determine their properties: including subcluster radii, intrinsic numbers of stars, central density, ellipticity, obscuration, and age. In 17 MYStIX regions we find 142 subclusters, with a diverse radii and densities and age spreads of up to ~1 Myr in a region. There is a strong negative correlation between subcluster radius and density, which indicates that embedded subclusters expand but also gain stars as they age. Subcluster expansion is also shown by a positive radius--age correlation, which indicates that subclusters are expanding at <1 km/s. The subcluster ellipticity distribution and number--density relation show signs of a hierarchical merger scenario, whereby young stellar clusters are built up through mergers of smaller clumps, causing evolution from a clumpy spatial distribution of stars (seen in some regions) to a simpler distribution of stars (seen in other regions). Many of the simple young stellar clusters show signs of dynamically relaxation, even though they are not old enough for this to have occurred through two-body interactions. However, this apparent contradiction might be explained if small subcluster, which have shorter dynamical relaxation times, can produce dynamically relaxed clusters through hierarchical mergers.

  11. Ares I-X Range Safety Analyses Overview

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Gowan, John W., Jr.; Thompson, Brian G.; Tarpley, Ashley W.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I Crew Launch Vehicle designed to provide manned access to low Earth orbit. As a one-time test flight, the Air Force's 45th Space Wing required a series of Range Safety analysis data products to be developed for the specified launch date and mission trajectory prior to granting flight approval on the Eastern Range. The range safety data package is required to ensure that the public, launch area, and launch complex personnel and resources are provided with an acceptable level of safety and that all aspects of prelaunch and launch operations adhere to applicable public laws. The analysis data products, defined in the Air Force Space Command Manual 91-710, Volume 2, consisted of a nominal trajectory, three sigma trajectory envelopes, stage impact footprints, acoustic intensity contours, trajectory turn angles resulting from potential vehicle malfunctions (including flight software failures), characterization of potential debris, and debris impact footprints. These data products were developed under the auspices of the Constellation's Program Launch Constellation Range Safety Panel and its Range Safety Trajectory Working Group with the intent of beginning the framework for the operational vehicle data products and providing programmatic review and oversight. A multi-center NASA team in conjunction with the 45th Space Wing, collaborated within the Trajectory Working Group forum to define the data product development processes, performed the analyses necessary to generate the data products, and performed independent verification and validation of the data products. This paper outlines the Range Safety data requirements and provides an overview of the processes established to develop both the data products and the individual analyses used to develop the data products, and it summarizes the results of the analyses required for the Ares I-X launch.

  12. Ares I-X Range Safety Flight Envelope Analysis

    NASA Technical Reports Server (NTRS)

    Starr, Brett R.; Olds, Aaron D.; Craig, Anthony S.

    2011-01-01

    Ares I-X was the first test flight of NASA's Constellation Program's Ares I Crew Launch Vehicle designed to provide manned access to low Earth orbit. As a one-time test flight, the Air Force's 45th Space Wing required a series of Range Safety analysis data products to be developed for the specified launch date and mission trajectory prior to granting flight approval on the Eastern Range. The range safety data package is required to ensure that the public, launch area, and launch complex personnel and resources are provided with an acceptable level of safety and that all aspects of prelaunch and launch operations adhere to applicable public laws. The analysis data products, defined in the Air Force Space Command Manual 91-710, Volume 2, consisted of a nominal trajectory, three sigma trajectory envelopes, stage impact footprints, acoustic intensity contours, trajectory turn angles resulting from potential vehicle malfunctions (including flight software failures), characterization of potential debris, and debris impact footprints. These data products were developed under the auspices of the Constellation's Program Launch Constellation Range Safety Panel and its Range Safety Trajectory Working Group with the intent of beginning the framework for the operational vehicle data products and providing programmatic review and oversight. A multi-center NASA team in conjunction with the 45th Space Wing, collaborated within the Trajectory Working Group forum to define the data product development processes, performed the analyses necessary to generate the data products, and performed independent verification and validation of the data products. This paper outlines the Range Safety data requirements and provides an overview of the processes established to develop both the data products and the individual analyses used to develop the data products, and it summarizes the results of the analyses required for the Ares I-X launch.

  13. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial.

    PubMed

    Santagostino, Elena; Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-04-07

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274.

  14. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

    PubMed Central

    Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-01-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

  15. Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients

    PubMed Central

    Negrier, Claude; Klamroth, Robert; Tiede, Andreas; Pabinger-Fasching, Ingrid; Voigt, Christine; Jacobs, Iris; Morfini, Massimo

    2012-01-01

    A recombinant fusion protein linking coagulation factor IX (FIX) with human albumin (rIX-FP) has been developed to facilitate hemophilia B treatment by less frequent FIX dosing. This first-in-human dose-escalation trial in 25 previously treated subjects with hemophilia B (FIX ≤ 2 IU/dL) examined the safety and pharmacokinetics of 25, 50, and 75 IU/kg rIX-FP. Patients in the 50-IU/kg cohort underwent a comparative pharmacokinetics assessment with their previous FIX product (plasma-derived or recombinant). No allergic reactions or inhibitors were observed. Four mild, possibly treatment-related adverse events were reported. In the 50-IU/kg cohort (13 subjects), the mean half-life of rIX-FP was 92 hours, more than 5 times longer than the subjects' previous FIX product. After 25 or 50 IU/kg rIX-FP administration, the baseline-corrected mean FIX activity remained elevated at day 7 (7.4 IU/dL and 13.4 IU/dL, respectively) and day 14 (2.5 IU/dL and 5.5 IU/dL, respectively). The incremental recovery of rIX-FP was higher than both recombinant and plasma-derived FIX (1.4 vs 0.95 and 1.1 IU/dL per IU/kg, respectively). These results demonstrated both the safety and improved pharmacokinetics of rIX-FP, thus indicating this new product with extended half-life as possibly able to control and prevent bleeding with less frequent injection. The trial was registered at www.clinicaltrials.gov as no. NCT01233440. PMID:22859609

  16. Characterization and standardization of tissue-simulating protoporphyrin IX optical phantoms

    NASA Astrophysics Data System (ADS)

    Marois, Mikael; Bravo, Jaime; Davis, Scott C.; Kanick, Stephen Chad

    2016-03-01

    Optical devices for measuring protoporphryin IX (PpIX) fluorescence in tissue are routinely validated by measurements in optical phantoms. Yet there exists limited data to form a consensus on the recipe for phantoms that both mimic the optical properties found in tissue and yield a reliable and stable relationship between PpIX concentration and the fluorescence remission intensity. This study characterizes the influence of multiple phantom components on PpIX fluorescence emission intensity, using Intralipid as the scattering source, bovine whole blood as the background absorber, and Tween as a surfactant to prevent PpIX aggregation. Optical measurements showed a linear proportionality (r>0.99) between fluorescence intensity and PpIX concentration (0.1 to 10 μg/mL) over a range of Intralipid (1 to 2%) and whole blood (0.5 to 3%) for phantoms containing low surfactant (≤0.1%), with fluorescence intensities and scattering and absorption properties stable for 5 h after mixing. The role of surfactant in PpIX phantoms was found to be complex, as aggregation was evident in aqueous nonturbid phantoms with no surfactant (0% Tween), and avoided in phantoms containing Intralipid as the scattering source with no additional or low amounts of added surfactant (≤0.1% Tween). Conversely, phantoms containing higher surfactant content (>0.1% Tween) and whole blood showed interactions that distorted the fluorescence emissions.

  17. Title IX and Pregnancy Discrimination in Higher Education: The New Frontier.

    PubMed

    Mason, Mary Ann; Younger, Jaclyn

    2014-01-01

    Pregnancy discrimination is a little known area covered by Title IX. According to the Title IX regulations, areas of prohibited discrimination include: admissions; hiring; coursework accommodations and completion; pregnancy leave policies and status protection upon return from leave; and health insurance coverage. These regulations will soon get more attention as the Obama Administration insists on Title IX dissemination and compliance in an effort to stop the leaky pipeline for women in the STEM fields. Research shows that pregnancy and childbirth are the major reasons why women drop out of research science in much greater numbers than men; this dropout is most likely to occur among graduate students and postdoctoral fellows who are in their peak childbearing years. A similar pattern of dropout can be seen in all fields, including related professional schools. Research also reveals that there are currently few established policies in higher education which adequately address pregnancy and childbirth in formal policies for students. This article will address new efforts by the United States Department of Education and the federal agencies to begin to seek compliance relating to Title IX and pregnancy discrimination in educational institutions. It will discuss the recent successful efforts of the U.S. Department of Education's Office for Civil Rights in investigating and settling pregnancy discrimination claims as well as the lessons learned in private action lawsuits under Title IX. Title IX private action suits have transformed athletics for women, and more recently Title IX has been applied in sexual harassment cases. Pregnancy discrimination is now the new frontier.

  18. Photosensitizing effect of hematoporphyrin IX on immature stages of Ceratitis capitata (Diptera: Tephritidae).

    PubMed

    Pujol-Lereis, Luciana Mercedes; Massaldi, Ana; Rabossi, Alejandro; Quesada-Allué, Luis Alberto

    2010-01-01

    Immature stages of Ceratitis capitata were tested as a model for hematoporphyrin IX (HP IX) phototoxicity. The lethal concentration 50 (LC(50)) of HP IX in the food was determined during postembryonic development until adult emergence as 0.173 mm (95% CI: 0.138-0.209). The corresponding HP IX LC(50) during the dispersal period alone was 0.536 mm (95% CI: 0.450-0.633). HP IX toxicity was compared against Phloxine B (PhB) (0.5 mm). HP IX elicited a mortality of 90.87%, which was mainly concentrated during prepupal and early pupal stages. PhB mortality was much lower (56.88%) and occurred mainly during the adult pharate stage. A direct correlation between light-dependent HP IX mortality, evidence of reactive oxygen species (ROS) and lipid peroxidation (conjugated dienes and thiobarbituric acid reactive substances) was established in C. capitata larvae. ROS were found to be very significant in both the brain and in the gut.

  19. Structural mechanics of the pH-dependent activity of beta-carbonic anhydrase from Mycobacterium tuberculosis.

    PubMed

    Covarrubias, Adrian Suarez; Bergfors, Terese; Jones, T Alwyn; Högbom, Martin

    2006-02-24

    Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. beta-carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a beta-carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a beta-carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.

  20. Ares I-X Flight Test Vehicle Similitude to the Ares I Crew Launch Vehicle

    NASA Technical Reports Server (NTRS)

    Huebner, Lawrence D.; Smith, R. Marshall; Campbell, John R.; Taylor, Terry L.

    2009-01-01

    The Ares I-X Flight Test Vehicle is the first in a series of flight test vehicles that will take the Ares I Crew Launch Vehicle design from development to operational capability. Ares I-X is scheduled for a 2009 flight date, early enough in the Ares I design and development process so that data obtained from the flight can impact the design of Ares I before its Critical Design Review. Decisions on Ares I-X scope, flight test objectives, and FTV fidelity were made prior to the Ares I systems requirements being baselined. This was necessary in order to achieve a development flight test to impact the Ares I design. Differences between the Ares I-X and the Ares I configurations are artifacts of formulating this experimental project at an early stage and the natural maturation of the Ares I design process. This paper describes the similarities and differences between the Ares I-X Flight Test Vehicle and the Ares I Crew Launch Vehicle. Areas of comparison include the outer mold line geometry, aerosciences, trajectory, structural modes, flight control architecture, separation sequence, and relevant element differences. Most of the outer mold line differences present between Ares I and Ares I-X are minor and will not have a significant effect on overall vehicle performance. The most significant impacts are related to the geometric differences in Orion Crew Exploration Vehicle at the forward end of the stack. These physical differences will cause differences in the flow physics in these areas. Even with these differences, the Ares I-X flight test is poised to meet all five primary objectives and six secondary objectives. Knowledge of what the Ares I-X flight test will provide in similitude to Ares I - as well as what the test will not provide - is important in the continued execution of the Ares I-X mission leading to its flight and the continued design and development of Ares I.