We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic Cterminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43-to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using f luorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with k cat /K m of 4.3 ؋ 10 7 M ؊1 s ؊1 , and k cat of 8.3 ؋ 10 4 s ؊1 . It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nM for acetazolamide, a clinically used sulfonamide, and of 0.25 M, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs.

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 M acetazolamide inhibited the relative invasion rate of these cell lines between 18 -74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells in vitro, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least in vitro, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.

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Carbonic anhydrase isozyme XII is a recently discovered member of the

Background: Carbonic anhydrase IX is a hypoxia-induced enzyme that has many biologically important functions, including its role in cell adhesion and invasion.

SUMMARY MN/CA IX is a recently discovered member of the carbonic anhydrase (CA) gene family that has been identified in the plasma membranes of certain tumor and epithelial cells and found to promote cell proliferation when transfected into NIH3T3 cells. This study presents localization of MN/CA IX in human gut and compares its distribution to those of CA I, II, and IV, which are known to be expressed in the intestinal epithelium. The specificity of the monoclonal antibody for MN/CA IX was confirmed by Western blots and immunostaining of COS-7 cells transfected with MN/CA IX cDNA. Immunohistochemical stainings of human gut revealed prominent polarized staining for MN/CA IX in the basolateral surfaces of the enterocytes of duodenum and jejunum, the reaction being most intense in the crypts. A moderate reaction was also seen in the crypts of ileal mucosa, whereas the staining became generally weaker in the large intestine. The results indicate isozyme-specific regulation of MN/CA IX expression along the cranial-caudal axis of the human gut and place the protein at the sites of rapid cell proliferation. The unique localization of MN/CA IX on the basolateral surfaces of proliferating crypt enterocytes suggests that it might serve as a ligand or a receptor for another protein that regulates intercellular communication or cell proliferation. Furthermore, MN/CA IX has a completely conserved active site domain of CAs suggesting that it could also participate in carbon dioxide/bicarbonate homeostasis. (J Histochem Cytochem 46:497-504, 1998)

Background During the COVID-19 pandemic, hospital staff have experienced a variety of mental health challenges. European research on anxiety and stress among hospital workers during the pandemic is limited. This study aimed to describe the anxiety levels of Finnish hospital workers during the COVID-19 pandemic. Methods The multidimensional, cross-sectional survey was distributed to all hospital staff working at two Finnish specialized medical care centres in the spring of 2020 ( n = 1,995). The Generalized Anxiety Disorder 7-item (GAD-7) scale was used to measure the workers’ anxiety. Results The total mean GAD-7 score was 4.88, indicating normal anxiety levels. However, 30% ( n =  1,079) of the respondents had mild, 10% ( n = 194) moderate and 5% ( n = 88) severe anxiety. Key risk factors were young age, working in a university hospital, problems in cooperation between co-workers, difficulty concentrating at work, a health-threatening physical and psychological workload, and a fear of being infected at work. Conclusion Hospital staff experienced a variety of work-related stress and anxiety issues that should be visible to hospital administrators and policymakers alike. The anxiety is independent of whether the worker is directly involved in caring for or in any way coming into contact with COVID-19 patients. Key message Fifty-five percent of hospital staff have normal anxiety levels. The remaining workers may need targeted support interventions, and a smaller proportion (15%) are in danger of developing longer-term problems affecting their well-being. The anxiety experienced by hospital workers during the COVID-19 pandemic is more severe than that of the population on average. If the pandemic continues, the well-being of hospital staff may be widely threatened. Despite the different geographical locations and COVID-19 situations, hospital workers in Finland and China had similar anxiety levels. The anxiety is independent of whether staff are working in the front line of managing the COVID-19 pandemic or of the number of covid-19 patients admitted to the hospital. The hospital workers felt anxiety because they were facing a new situation which causes changes in their work and daily routine. Health care employers should engage in long-term follow-up as regards the personnel’s recovery from the burden caused by the pandemic and from work in general. It is necessary to make easily attainable, flexibly delivered and cost-effective treatment interventions for anxiety available to hospital staff.