MN/CA IX/G250 as a potential target for immunotherapy of renal cell carcinomas.
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The monoclonal antibody G250 (mAbG250) raised against a human renal cell carcinoma (RCC) has been shown to react with a large number of RCCs. Recently, G250 antigen was isolated and found to be homologous to the MN/CA9 gene originally identified in HeLa cells. To determine whether G250 antigen (MN/CA IX/G250) could be a potential therapeutic target and a tumour marker, a total of 147 cases of RCC were investigated immunohistochemically as well as by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. In addition, total RNAs extracted from patients' peripheral blood samples were analysed for MN/CA9/G250 mRNA signals. Immunohistochemistry demonstrated strong expression in 128/147 (87.1%) of RCCs, in contrast to the lack of expression observed in normal tissues. RT-PCR analyses of frozen specimens resulted in the clear detection of MN/CA9/G250 mRNA signals in 137/147 (93.2%), and despite subtle differences the results were almost identical to those for immunohistochemistry. Although high-grade and -stage tumours exhibited significantly lower expression than low-grade and -stage tumours, a large proportion of tumours expressed MN/G250 protein as well as mRNA. RT-PCR analysis of patients' blood samples revealed the presence of circulating MN/CA9/G250 expressing cells. These findings suggest that this antigen may be a potential therapeutic target as well as diagnostic marker for RCCs. (+info)
CO(2)-responsive transcriptional regulation of CAH1 encoding carbonic anhydrase is mediated by enhancer and silencer regions in Chlamydomonas reinhardtii.
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Chlamydomonas reinhardtii adapts to the stress of CO(2)-limiting conditions through the induction of a set of genes including CAH1, which encodes a periplasmic carbonic anhydrase. CAH1 is up-regulated under low-CO(2) conditions (air containing 0.04% [v/v] CO(2)) in the presence of light, whereas it is down-regulated under high-CO(2) conditions (5% [v/v] CO(2)) or in the dark. In an effort to identify cis-elements involved in the transcriptional regulation of CAH1, a series of 5'-nested deletions of the region upstream of CAH1 were fused to a promoterless arylsulfatase reporter gene (ARS). The upstream region from -651 to +41 relative to the transcription start site was sufficient to regulate the expression of ARS with kinetics similar to those of endogenous CAH1. Deletion of the region between -651 and -294 resulted in a significant decrease in the level of arylsulfatase activity expressed under low-CO(2) conditions. The 543-bp upstream region from -651 to -109, without any promoter elements, CAAT-box, or TATA-box, could confer CO(2) and light responsiveness on the beta(2)-tubulin minimal promoter. This 543-bp region was divided into two parts: a 358-bp silencer region from -651 to -294, which represses the minimal promoter activity under high-CO(2) conditions, and a 185-bp enhancer region from -293 to -109, which activates the promoter under low-CO(2) conditions in the presence of light. (+info)
Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.
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Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergent-solubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle. (+info)
Carbonic anhydrase is an ancient enzyme widespread in prokaryotes.
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Carbonic anhydrases catalyze the reversible hydration of CO(2) and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (gamma class) in a methanoarchaeon and our present finding that the beta class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the beta and gamma classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized. (+info)
Characterization of multipole storage assisted dissociation: implications for electrospray ionization mass spectrometry characterization of biomolecules.
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Ions accumulated in an rf-only multipole for extended periods of time prior to mass analysis can experience a significant degree of fragmentation and produce mass spectra which do not reflect the true nature of the intact analyte(s). This phenomenon, termed multipole storage assisted dissociation (MSAD), places constraints on the maximum number of ions which can be accumulated in the multipole storage device as a result of its finite space charge limit. This phenomenon can be exploited to produce dissociation spectra that are dominated by fragment ions providing important sequence/structure information. In this work we further explore MSAD and characterize parameters including accumulation time, source pressure, and the electrostatic configuration of the multipole storage device, which mediate the phenomenon. Operating parameters are identified that can either enhance or eliminate the phenomenon. (+info)
Carbon metabolism in developing soybean root nodules: the role of carbonic anhydrase.
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A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development. (+info)
Carbonic anhydrase inhibition delays plasma lactate appearance with no effect on ventilatory threshold.
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The effect of carbonic anhydrase (CA) inhibition with acetazolamide (Acz, 10 mg/kg body wt iv) on exercise performance and the ventilatory (VET) and lactate (LaT) thresholds was studied in seven men during ramp exercise (25 W/min) to exhaustion. Breath-by-breath measurements of gas exchange were obtained. Arterialized venous blood was sampled from a dorsal hand vein and analyzed for plasma pH, PCO(2), and lactate concentration ([La(-)](pl)). VET [expressed as O(2) uptake (VO(2)), ml/min] was determined using the V-slope method. LaT (expressed as VO(2), ml/min) was determined from the work rate (WR) at which [La(-)](pl) increased 1.0 mM above rest levels. Peak WR was higher in control (Con) than in Acz sutdies [339 +/- 14 vs. 315 +/- 14 (SE) W]. Submaximal exercise VO(2) was similar in Acz and Con; the lower VO(2) at exhaustion in Acz than in Con (3.824 +/- 0. 150 vs. 4.283 +/- 0.148 l/min) was appropriate for the lower WR. CO(2) output (VCO(2)) was lower in Acz than in Con at exercise intensities >/=125 W and at exhaustion (4.375 +/- 0.158 vs. 5.235 +/- 0.148 l/min). [La(-)](pl) was lower in Acz than in Con during submaximal exercise >/=150 W and at exhaustion (7.5 +/- 1.1 vs. 11.5 +/- 1.1 mmol/l). VET was similar in Acz and Con (2.483 +/- 0.086 and 2.362 +/- 0.110 l/min, respectively), whereas the LaT occurred at a higher VO(2) in Acz than in Con (2.738 +/- 0.223 vs. 2.190 +/- 0.235 l/min). CA inhibition with Acz is associated with impaired elimination of CO(2) during the non-steady-state condition of ramp exercise. The similarity in VET in Con and Acz suggests that La(-) production is similar between conditions but La(-) appearance in plasma is reduced and/or La(-) uptake by other tissues is enhanced after the Acz treatment. (+info)
Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors.
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Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior. (+info)