Radioimmunoassay of carbonic anhydrase III in rat tissues.
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ABSTRACT:
SUBMITTER: Shiels A
PROVIDER: S-EPMC1153339 | biostudies-other | 1984 Mar
REPOSITORIES: biostudies-other
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Project description:A specific and sensitive radioimmunoassay has been developed for the measurement of the secreted carbonic anhydrase isoenzyme (CA VI) in sheep saliva and tissues. The assay can detect as little as 75 pg of CA VI, and the antibody used does not cross-react with CA II or CA III. The intra-assay variation, measured using a saliva sample, was 3.0%, whereas the inter-assay variation was 10.5%. The concentration of CA VI in parotid saliva from normal, resting sheep was 5.6 +/- 3.0 micrograms.ml-1 (n = 42) or 79.4 +/- 35.7 micrograms.mg of total protein-1. With feeding, the CA VI concentrations increased an average of 6-fold. The secretion rate of CA VI from the vascularly isolated neurotomized parotid gland of the anaesthetized sheep was 0.62 +/- 0.40 micrograms.min-1, compared with a rate of 11.7 +/- 7.8 micrograms.min-1 from the parotid gland of normal conscious sheep. Stimulation of the parotid-gland preparation by the muscarinic agent bethanechol increased the secretion rate to 438 +/- 172 microgram.min-1 (n = 8), and electrical stimulation of the secretomotor Moussu nerve increased CA VI secretion rate to 634 +/- 330 micrograms.min-1 (n = 4). Submandibular saliva from anaesthetized sheep contained 6.9 +/- 2.1 micrograms of CA VI.ml-1 (n = 3). The only tissues found to contain measurable amounts of CA VI were the parotid (6.4 micrograms.mg of protein-1) and submandibular (1.8 micrograms.mg of protein-1) salivary glands. The sublingual salivary gland, kidney, lung, adrenal, brain, skeletal muscle, liver, heart, pancreas, small intestine and cerebrospinal fluid did not have a measurable CA VI content.
| S-EPMC1150138 | biostudies-other
Project description:Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.
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Project description: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(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(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-?2 (PPAR?2) and CCAAT/enhancer binding protein-?. We found a considerable (approximately 1000-fold) increase in the PPAR?2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPAR?2 and FABP4. When both CAIII and PPAR?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?2 gene expression.
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Project description:Carbonic anhydrase III (CAIII) protein and mRNA amounts in fast- and slow-twitch rat muscles were examined after resection of the sciatic nerve. Striking changes occur in the fast-twitch anterior tibialis (AT) and extensor digitorum longus (EDL) muscles, where CAIII protein and mRNA are increased several-fold 16 days after denervation. The data suggest that these changes are regulated in part by changes in gene transcription and that they perhaps signal a fast-to-slow fibre type transition in these denervated muscles. AT and EDL show some differences in the effects of denervation, which are suggestive of variation in the timing of denervation-induced responses and/or the CAIII protein/mRNA turnover rates in the two muscles.
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Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction
2012-03-25 | GSE36625 | GEO
Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction 6 dye-swap - gene knock out
2012-03-25 | E-GEOD-36625 | biostudies-arrayexpress
Project description:We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ? 75-80 kDa, ? 95-100 kDa, and ? 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ? 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (? 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.
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Project description:Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.
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Project description:Epithelial-mesenchymal transition (EMT) is strongly correlated with tumor metastasis and contains several protein markers, such as E-cadherin. Carbonic anhydrase III (CA III) exhibits low carbon dioxide hydratase activity in cancer. However, the detailed mechanisms of CA III and their roles in oral cancer are still unknown. This study established a CA III-overexpressed stable clone and observed the expression of CA III protein in human SCC-9 and SAS oral cancer cell lines. The migration and invasion abilities were determined using a Boyden chamber assay. Our results showed that the overexpression of CA III protein significantly increased the migration and invasion abilities in oral cancer cells. Moreover, a whole genome array analysis revealed that CA III regulated epithelial-mesenchymal transition by reducing the expression of epithelial markers. Data from the GEO database also demonstrated that CA III mRNA is negatively correlated with CDH1 mRNA. Mechanistically, CA III increased the cell motility of oral cancer cells through the FAK/Src signaling pathway. In conclusion, this suggests that CA III promotes EMT and cell migration and is potentially related to the FAK/Src signaling pathway in oral cancer.
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Project description:The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.
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