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Aim: To describe hospital staff's experiences of management actions to promote their mental well-being during the COVID-19 pandemic. Mental well-being was examined on the basis of four entities: level of anxiety, support and encouragement from the manager, and the opportunity to discuss concerns about COVID-19 with the manager., Background: The workload of COVID-19 affects the mental well-being of staff. However, there is limited data on managers' actions to promote their mental well-being during the pandemic., Methods: A cross-sectional study was used to collect survey data (n = 1995) among staff working in two specialized medical care hospitals. To gain deeper understanding related issues, the survey included open questions, which were answered by 178 participants., Results: The results indicate that those staff who felt they had received support, encouragement, and the opportunity to discuss of COVID-19 worries with a manager experienced less anxiety., Conclusions: The study provides an insight into managers' actions to promote staff's mental well-being during the COVID-19 pandemic., Implications for Nursing Management: The manager's actions have a significant effect on the anxiety levels of staff. During the pandemic, the well-being of staff is a priority that should be visible to both hospital administrators and policymakers., (© 2022 John Wiley & Sons Ltd.)

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.

Objectives: To examine the occurrence of and recovery from visual neglect-related symptoms with the focus on neglect laterality, ipsilateral orienting bias, and slowed processing speed in right hemisphere (RH) infarct patients during a 1-year follow-up. Furthermore, to propose guidelines for assessing processing speed alongside the Behavioural Inattention Test (BIT)., Methods: We studied three RH patient groups: neglect (N+), mild left inattention (MLI+), and non-neglect (N-) patients, and healthy controls. The BIT with some additional analyses was conducted at the acute phase and at 6 and 12 months., Results: The N+ group's BIT score increased and originally lateralized omissions became more evenly distributed during the follow-up. The N+ and MLI+ groups' starting points were more rightward located than the healthy group's at the acute phase and at 6, and partly at 12 months. Patient groups were slower than the controls in performing cancellation tests at the acute phase. The N+ and MLI+ groups remained slower than the controls throughout the follow-up., Conclusions: During the first year after RH infarct, originally left-sided manifestation of neglect shifted toward milder non-lateralized attentional deficit. Ipsilateral orienting bias and slowed processing speed appeared to be rather persistent neglect-related symptoms both in neglect patients and patients with initially milder inattention. We propose some effortless, tentative ways of examining processing speed and ipsilateral orienting bias alongside the BIT to better recognize these neglect-related symptoms, and highlight the need to assess and treat patients with initially milder inattention, who have been under-recognized and under-treated in clinical work. (JINS, 2018, 24, 617-628).

Objective: This prospective follow-up study aimed to identify sociodemographic and clinical factors that may affect the quality of life (QoL) of patients with acute ischemic stroke during a 6-month follow-up., Patients and Methods: In the acute phase, sociodemographic and clinical data were collected using the National Institute of Health Stroke Scale, Barthel Index, and modified Rankin Scale. QoL was assessed with the Stroke and Aphasia Quality of Life Scale-39 6 months after stroke., Results: QoL was evaluated in 64 patients (aged 45-81 years) with a first-ever ischemic stroke. Thrombolytic therapy was given to 80% of the patients. Stroke severity, dependence in activities of daily living, degree of handicap, and length of hospitalization were associated with QoL. QoL was not associated with age, gender, marital status, or years of education., Conclusion: In this study, most patients were treated with thrombolysis, and QoL results resembled those of earlier studies on patients without thrombolysis. Despite good physical recovery, the patients reported impairments in QoL. QoL assessments can give clinicians a more holistic picture of stroke recovery from the patient's perspective., (© 2016 S. Karger AG, Basel.)

Background and Purpose: Several prognostic scores have been developed to predict the risk of symptomatic intracranial hemorrhage (sICH) after ischemic stroke thrombolysis. We compared the performance of these scores in a multicenter cohort., Methods: We merged prospectively collected data of patients with consecutive ischemic stroke who received intravenous thrombolysis in 7 stroke centers. We identified and evaluated 6 scores that can provide an estimate of the risk of sICH in hyperacute settings: MSS (Multicenter Stroke Survey); HAT (Hemorrhage After Thrombolysis); SEDAN (blood sugar, early infarct signs, [hyper]dense cerebral artery sign, age, NIH Stroke Scale); GRASPS (glucose at presentation, race [Asian], age, sex [male], systolic blood pressure at presentation, and severity of stroke at presentation [NIH Stroke Scale]); SITS (Safe Implementation of Thrombolysis in Stroke); and SPAN (stroke prognostication using age and NIH Stroke Scale)-100 positive index. We included only patients with available variables for all scores. We calculated the area under the receiver operating characteristic curve (AUC-ROC) and also performed logistic regression and the Hosmer-Lemeshow test., Results: The final cohort comprised 3012 eligible patients, of whom 221 (7.3%) had sICH per National Institute of Neurological Disorders and Stroke, 141 (4.7%) per European Cooperative Acute Stroke Study II, and 86 (2.9%) per Safe Implementation of Thrombolysis in Stroke criteria. The performance of the scores assessed with AUC-ROC for predicting European Cooperative Acute Stroke Study II sICH was: MSS, 0.63 (95% confidence interval, 0.58-0.68); HAT, 0.65 (0.60-0.70); SEDAN, 0.70 (0.66-0.73); GRASPS, 0.67 (0.62-0.72); SITS, 0.64 (0.59-0.69); and SPAN-100 positive index, 0.56 (0.50-0.61). SEDAN had significantly higher AUC-ROC values compared with all other scores, except for GRASPS where the difference was nonsignificant. SPAN-100 performed significantly worse compared with other scores. The discriminative ranking of the scores was the same for the National Institute of Neurological Disorders and Stroke, and Safe Implementation of Thrombolysis in Stroke definitions, with SEDAN performing best, GRASPS second, and SPAN-100 worst., Conclusions: SPAN-100 had the worst predictive power, and SEDAN constantly the highest predictive power. However, none of the scores had better than moderate performance.

Background and Purpose: Inverse relationship between onset-to-door time (ODT) and door-to-needle time (DNT) in stroke thrombolysis was reported from various registries. We analyzed this relationship and other determinants of DNT in dedicated stroke centers., Methods: Prospectively collected data of consecutive ischemic stroke patients from 10 centers who received IV thrombolysis within 4.5 hours from symptom onset were merged (n=7106). DNT was analyzed as a function of demographic and prehospital variables using regression analyses, and change over time was considered., Results: In 6348 eligible patients with known treatment delays, median DNT was 42 minutes and kept decreasing steeply every year (P<0.001). Median DNT of 55 minutes was observed in patients with ODT ≤30 minutes, whereas it declined for patients presenting within the last 30 minutes of the 3-hour time window (median, 33 minutes) and of the 4.5-hour time window (20 minutes). For ODT within the first 30 minutes of the extended time window (181-210 minutes), DNT increased to 42 minutes. DNT was stable for ODT for 30 to 150 minutes (40-45 minutes). We found a weak inverse overall correlation between ODT and DNT (R(2)=-0.12; P<0.001), but it was strong in patients treated between 3 and 4.5 hours (R(2)=-0.75; P<0.001). ODT was independently inversely associated with DNT (P<0.001) in regression analysis. Octogenarians and women tended to have longer DNT., Conclusions: DNT was decreasing steeply over the last years in dedicated stroke centers; however, significant oscillations of in-hospital treatment delays occurred at both ends of the time window. This suggests that further improvements can be achieved, particularly in the elderly.

Preliminary observations have suggested mild behavioral changes and a morphological disruption of brain histology in 1.5-year-old carbonic anhydrase IX (CA IX)-deficient (Car9 (-/-)) mice. These findings led us to design a 1-year follow-up study in which the behavior and brain histology of Car9 (-/-) and wild-type mice were monitored. Morphological analysis revealed vacuolar degenerative changes in the brains of Car9 (-/-) mice. The changes became visible at the age of eight to ten months. Behavioral tests showed that the Car9 (-/-) mice exhibited abnormal locomotor activity and poor performance in a memory test. To further identify the transcriptomic responses to CA IX deficiency in the brain, genome-wide cDNA microarray analyses were performed. Thirty-one and 37 genes were significantly up- or down-regulated, respectively, in the brain of Car9 (-/-) mice compared to the wild-type mice. Functional annotation revealed that the genes with increased expression were involved in several processes, such as RNA metabolism, and the genes with reduced expression were implicated in other important processes, including the regulation of cellular ion homeostasis. Notably, the biological processes "behavior" and "locomotory behavior" were the two prominent terms overrepresented among the down-regulated genes, which is consistent with the behavioral phenotype. These results suggest that CA IX may directly or indirectly play novel functions in brain tissue. Furthermore, the brain phenotype of Car9 (-/-) mice seems to be age-dependent. The results indicate that the functional changes precede the microscopic alterations in the brains of Car9 (-/-) mice.

Gastrointestinal stromal tumors (GISTs) are clinically distinct mesenchymal tumors, which generally result from expression of mutant KIT or PDGFRA receptor tyrosine kinase oncogenes. Most GISTs feature strong expression of KIT that serves as a crucial diagnostic adjunct. However, a subset of tumors lacks KIT expression and otherwise may also be difficult to distinguish from other sarcomas, including leiomyosarcoma. Because various carbonic anhydrase (CA) isozymes have been identified as potential treatment targets against different cancers, we evaluated CA II expression in 175 GISTs. Western blotting experiments indicated that CA II is highly expressed in GIST cell lines. Immunohistochemically, 95% of GISTs showed positive signal. The CA II expression in GISTs did not correlate with particular KIT or PDGFRA mutation types. CA II immunoreactivity was absent or low in other mesenchymal tumor categories analyzed. High CA II expression was associated with a better disease-specific survival rate than low or no expression (Mantel-Cox test, P<0.0001). The present results indicate that CA II is overexpressed in most GISTs, is quite selective to this tumor type among mesenchymal tumors, and therefore might be a useful biomarker in diagnostics.

Background: Medulloblastomas (MBs) and supratentorial primitive neuroectodermal tumours (PNETs) are the most common highly aggressive paediatric brain tumours. In spite of extensive research on these tumours, there are only few known biomarkers or therapeutic target proteins, and the prognosis of patients with these tumours remains poor. Our aim was to investigate whether carbonic anhydrases (CAs), enzymes commonly overexpressed in various tumours including glioblastomas and oligodendrogliomas, are present in MBs and PNETs, and whether their expression can be correlated with patient prognosis., Methods: We determined the expression of the tumour-associated carbonic anhydrases CA II, CA IX and CA XII in a series of MB/PNET specimens (n = 39) using immunohistochemistry., Results: Endothelial CA II, cytoplasmic CA II, CA IX and CA XII were expressed in 49%, 73%, 23% and 11% of the tumours, respectively. CA II was detected in the neovessel endothelium and the tumour cell cytoplasm. CA IX was mainly expressed in the tumour cells located in perinecrotic areas. CA XII showed the most homogenous distribution within the tumours. Importantly, CA IX expression predicted poor prognosis in both univariate (p = 0.041) and multivariate analyses (p = 0.016)., Conclusions: We suggest that CA IX should be considered a potential prognostic and therapeutic target in MBs and PNETs.

Object: Carbonic anhydrase (CA) II and IX are enzymes involved in pH homeostasis and have been shown to be upregulated in several types of cancer. In this study, the authors evaluate the expression of CA II and IX in meningiomas and assess their relationship to patient age, tumor type and grade, tumor sex hormone receptor status, tumor cell proliferation, and tumor recurrence., Methods: This study was conducted in consecutive patients who underwent meningioma surgeries at Tampere University Hospital between 1989 and 1999. The expression of CA II and IX was studied immunohistochemically using a tissue microarray technique and specific antibodies., Results: Immunohistological staining with CA II and IX was assessed in 443 primary and 67 recurrent tumor specimens. Of these samples, 455 were benign (WHO Grade I), 49 atypical (Grade II), and 6 malignant (Grade III). Endothelial cells in 14.8% of the tumors stained positively for CA II. Tumor cells were positive for CA IX in 11.6% of the cases. Endothelial CA II expression correlated with increasing histological grade (p=0.002), and tumor proliferation rates were higher in CA II+ versus CA II- cases (p=0.002). Androgen receptor-negative tumors were found to be CA II+ significantly more often than androgen receptor-positive tumors (p=0.001). No associations were found with the CA IX enzyme., Conclusions: Carbonic anhydrase II positivity in the endothelium was associated with cell proliferation and malignancy grade. These results suggest that CA II expression is associated with malignant progression of meningiomas and could thus be a target molecule for anticancer therapy.

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.

Background: Carbonic anhydrase IX is a hypoxia-induced enzyme that has many biologically important functions, including its role in cell adhesion and invasion., Methods: This study was set out to investigate the role of CA IX in a series of 86 oligodendroglial brain tumors (71 primary and 15 recurrent; 48 pure oligodendrogliomas and 40 mixed oligoastrocytomas)., Results: 80% of the tumors showed CA IX expression by immunohistochemistry. Tumors with moderate or strong CA IX expression had decreased level of cell proliferation compared to weak or no CA IX expression (median 2.9 vs. 5.8, p = 0.015). CA IX correlated with two antioxidative enzymes, manganese superoxide dismutase (MnSOD) and regulatory gammaglutamylcysteine synthetase (GLCL-R): CA IX expression was significantly higher in MnSOD-positive tumors (p = 0.008) and decreased in GLCL-R-positive tumors (p = 0.044). In Cox multivariate analysis CA IX expression, patient age and histological component (pure oligodendroglioma vs. mixed oligoastrocytoma) showed independent prognostic values (p = 0.009, p = 0.003 and p = 0.022, respectively), CA IX positivity predicting poorer outcome., Conclusion: CA IX was proved to be an independent prognostic indicator in oligodendroglial brain tumors, and it also correlates reversely with cell proliferation. It may have a role in the biology of oligodendrogliomas, and most interestingly, as it is mainly expressed in tumor tissue, CA IX could serve as a target molecule for anticancer treatments.

Carbonic anhydrase (CA) II, CA IX, and CA XII are expressed in various neoplasias and have been linked to tumorigenesis. We examined their expression in three different groups of colorectal cancer [i.e., microsatellite stable (MSS), microsatellite instable (MSI), and hereditary nonpolyposis colorectal cancer (HNPCC)]. First, we analyzed gene expression profiles of 113 specimens by a microarray method to study the expression of various CA isozymes in the subgroups of colorectal cancer. The results indicated that mRNAs for CA II and CA XII are down-regulated and CA IX mRNA is up-regulated in all three tumor categories when compared with the normal tissue. The up-regulation of CA IX was greatest in the HNPCC group. For more information, 77 specimens were immunohistochemically stained to study the levels of CA II, CA IX, and CA XII. Immunohistochemical analyses further confirmed that the subgroups express CA II, CA IX, and CA XII differentially, and the HNPCC tumors express high levels of CA IX. Expression of these CAs did not correlate to Dukes stage or grade of differentiation. Our results show that CAs are differentially expressed in the subgroups of colorectal cancer, and CA IX expression seems to be very high in most cases of HNPCC. CA IX could be a potential diagnostic and therapeutic target in HNPCC.

Carbonic anhydrase isozyme II (CA II) is a cytosolic enzyme that is highly expressed in most organs, including the brain, where it is mainly located in the oligodendrocytes. Recent studies have shown that its expression is induced in the endothelium of neovessels in melanoma and esophageal, renal, and lung cancer. Immunological studies further indicate that CA II represents a major target antigen stimulating an autoantibody response in melanoma patients. These results prompted us to investigate endothelial CA II expression in two types of brain cancer: oligodendrogliomas and astrocytomas. A series of 255 astrocytoma and 71 oligodendroglial tumor specimens was immunostained for CA II. The staining results were correlated with a number of different clinicopathological factors and survival data. CA II showed weak or no expression in low-grade tumors, while grade 3 mixed oligoastrocytoma and glioblastoma multiforme were the most positively stained tumor types. Survival analysis indicated that endothelial CA II staining is significantly associated with a poor prognosis in patients with astrocytomas. About 17% of patients with CA II-negative tumors (weak or no endothelial signal) were still alive at the end of the follow-up period of five years. The presence of CA II in the tumor endothelium suggests that it may play an important functional role in tumor metabolism. From a clinical perspective, the results also open new avenues for selecting tumor types for dendritic cell therapy trials.

The first activation study of isoform XIII of carbonic anhydrase (CA, EC 4.2.1.1) is reported. A series of amino acids and amines incorporating protonatable moieties of the primary/heterocyclic amine type were included in the study. As for CA I and II, CA XIII activators enhance kcat and show no effect on KM, for the physiologic reaction catalyzed by this isoform. Excellent CA XIII activating properties were shown by D-amino acids (His, Phe, DOPA, and Trp), serotonin, and 4-(2-aminoethyl)-morpholine, whereas the corresponding L-amino acids, dopamine, histamine, and 1-(2-aminoethyl)-piperazine, were weaker activators.

Purpose: Carbonic anhydrase IX (CA IX) is a hypoxia-inducible enzyme, which is associated with neoplastic growth. Ectopic CA IX expression has been observed in several tumors, whose normal counterparts do not express this enzyme. Normal human brain tissue shows only slight or no expression of CA IX., Experimental Design: We describe CA IX expression in human diffusely infiltrating astrocytomas. The association of CA IX is evaluated with clinicopathologic and molecular factors including cell proliferation and apoptosis as well as the expression of p53 and epidermal growth factor receptor., Results: CA IX immunopositivity was observed in 284 cases of 362 (78%) tumors. The positive areas were often located in close proximity to necrotic regions (P < 0.001). The CA IX immunoreactivity showed strong association with tumor malignancy grades (P < 0.0001). CA IX showed no association with p53 expression nor did it correlate with epidermal growth factor receptor-amplification, apoptosis, or cell proliferation. CA IX intensity had significant prognostic value in univariate (P=0.0011, log-rank test) and multivariate survival analysis (P = 0.038, Cox analysis)., Conclusions: CA IX expression is common in diffusely infiltrating high-grade astrocytomas. Our results suggest that CA IX is a useful biomarker for predicting poor prognosis of astrocytic tumors. It may also be a promising target molecule for the improvement of therapeutic interventions in astrocytomas.

Aim: To analyze possible relationships between CA IX/CA XII and pVHL expression in normal and neoplastic colorectal mucosa., Methods: Immunohistochemical staining of 42 tissue specimens obtained from 17 cancer patients was performed to evaluate the distribution and semi-quantitatively assess the levels of CA IX, CA XII and pVHL. VHL mRNAs from 14 fresh-frozen tumors was amplified by RT-PCR and subjected to sequencing. CA9 and CA12 mRNA levels were analyzed by semi-quantitative RT-PCR in comparison with VEGF as an indicator of hypoxia that uncouples the pVHL control., Results: Tumor tissues were associated with a borderline increase of CA IX staining signal and slight but significant decrease of CA XII immunoreactivity, whereas no association was found for pVHL. Sequence analysis of RT-PCR-amplified VHL mRNAs revealed no deletions/mutations, suggesting that they were VHL-competent. We did not observe any correlation between pVHL and CA IX/CA XII proteins as well as between VEGF and CA9 mRNAs, but the tumor-associated changes in mRNA levels of VEGF and CA12 showed a significant inverse relationship., Conclusion: Our results indicate that CA9 and CA12 are regulated by different intratumoral factors and that lack of apparent relationship between the levels of CA IX/CA XII and pVHL cannot be fully assigned to uncoupling of negative regulatory function of pVHL by tumor hypoxia signified by induced VEGF transcription. The interplay between the functional pVHL and CA IX/CA XII in colorectal tumors seems rather complex and is not evident merely at the expression levels.

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 x 10(7) m(-1) s(-1), and k(cat) of 8.3 x 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 microm, 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.

Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure-function relationships in the CA IX molecule and a better understanding of the role of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA IX detection in body fluids of cancer patients.

Background: Hereditary hemochromatosis (HH), a disease involving iron accumulation in internal organs, occurs in about 1 in 200-400 Caucasians. The gene mutated in this disorder is termed HFE. The present study was designed to evaluate the diagnostic utility and outcome of genetic testing for HH in the service of public health care., Methods: 137 subjects were referred by health clinics and general hospitals for HFE genotyping from various parts of Finland during the period 1999-2001. Two major mutations (C282Y and H63D) were determined for each patient. Reasons contributing to referrals and sets of values for serum transferrin saturation (s-TS) and iron and ferritin concentrations were also determined., Results: 16.8% of the subjects were homozygous for the C282Y mutation, together with seven C282Y/H63D compound heterozygotes (5.1%). The rate of positive findings for the most typical mutations responsible for HH was found to have increased steadily during the period 1999-2001., Conclusions: Our data support a role for active testing for the C282Y and H63D mutations in health care. The fairly low number of genotyping requests nevertheless suggests that a large number of patients even with typical clinical signs or symptoms continue to escape detection.

Purpose: Carbonic anhydrases (CAs) are key enzymes that regulate acid-base homeostasis in both normal and pathological conditions. Recent studies have shown that they are functionally involved in the growth and invasion of cancer cells. However, there are only a few publications on CAs in hematological malignancies., Experimental Design: Here we investigated the presence of CA isozymes in six malignant hematopoietic cell lines and malignant blast cells of bone marrow samples collected from patients with acute myeloid leukemia, acute lymphoblastic leukemia, or chronic myelomonocytic leukemia., Results: Because three of the malignant hematopoietic cell lines expressed CA II, we also set out to examine its expression in a series of bone marrow samples. Positive reactions were found in 16 of 26 cases (62%) of acute myeloid leukemia, 11 of 15 cases (73%) of acute lymphoblastic leukemia, and 1 of 2 cases (50%) of chronic myelomonocytic leukemia., Conclusions: The results indicate that CA II expression is not restricted to only one cell lineage but may result from a genetic aberration that occurs in both myeloid and lymphatic lineages or in their progenitor cell. Because CA II is expressed in most patients with leukemic blast cells, CA inhibitors may prove to be of value as an adjunct to chemotherapy for such cancers.

Background: Biochemical and histochemical studies have both previously indicated plasma membrane-associated carbonic anhydrase (CA) activity in hepatocytes which has been assumed to be CA IV. However, immunohistochemical data did not support this assignment. Recent northern blotting results indicated the presence of mRNA for the most recently discovered membrane-bound CA isozyme, CA XIV, in the liver. The present study was designed to examine whether CA XIV could contribute to the CA activity described in the hepatocytes., Methods: Tissue samples from mouse liver were subjected to immunohistochemical staining using the antibodies raised against recombinant mouse CA XIV and CA IV. RT-PCR and western blotting were also performed for CA XIV., Results: A strong immunofluorescent signal was observed in the plasma membrane of mouse hepatocytes. Although CA XIV was expressed on both the apical and basolateral surfaces, the staining was more prominent at the apical (canalicular) membrane domain. The expression of CA XIV in the liver was confirmed by RT-PCR and western blotting., Conclusions: The presence of CA XIV in the hepatocyte plasma membrane places this novel enzyme at a strategic site to control pH regulation and ion transport between the hepatocytes, sinusoids and bile canaliculi.

Background/aims: Carbonic anhydrase isoenzyme IX (MN/CA IX) is a transmembrane protein with a suggested function in maintaining the acid-base balance and intercellular communication. Previous studies have demonstrated that MN/CA IX is expressed in the basolateral plasma membrane of normal biliary epithelial cells, but not in hepatocytes. This study was designed to examine the expression of MN/CA IX in hepatobiliary neoplasms and to elucidate its value as a marker for biliary differentiation., Methods: Fifty-seven hepatobiliary lesions were immunostained for MN/CA IX using biotin-streptavidin complex method. Twenty samples containing normal biliary epithelium and five containing normal liver tissue were used as controls., Results: In the biliary epithelial tumours, immunostaining for MN/CA IX was mainly localized at the basolateral surface of the epithelial cells, like in normal mucosa. All non-invasive dysplastic lesions and 57% of invasive lesions of gall-bladder expressed MN/CA IX. In liver, 78% of cholangiocellular malignant lesions showed a positive reaction for MN/CA IX, whereas only 33% of hepatocellular carcinomas showed a weak immunoreaction., Conclusions: Our results suggest that abnormal expression of MN/CA IX may be linked to malignant transformation of hepatobiliary cells. In addition, it seems to be a promising marker for biliary differentiation in hepatobiliary neoplasms.

This study compares the localization of carbonic anhydrase isozymes (CA) I and II and that of IX and XII in normal large intestine and in colorectal tumors. Immunohistochemical studies were performed on 69 colorectal lesions. While the normal mucosa of the large intestine showed high expression for CA I and II, the intensity of the immunostaining for both isozymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pattern of expression observed for these cytoplasmic isozymes and transmembrane CA IX and XII in intestinal tissue specimens supports the suggestion that CA IX and XII may be functionally involved in tumor progression to malignancy and/or in invasion. By contrast, while CA I and II are prominent in normal colorectal mucosa, where they play a role in regulation of pH homeostasis and water and ion transport, loss of expression of these cytoplasmic isozymes consistently accompanies progression to malignant transformation.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.

Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes which are expressed in several epithelia and overexpressed in some carcinomas. They have recently been linked to von Hippel-Lindau gene-mediated carcinogenesis in that both isoenzymes are downregulated by the product of the wild-type von Hippel-Lindau tumour suppressor gene. This paper describes the localisation of CA IX and XII in the normal human pancreas and pancreatic tumours. Both isoenzymes showed positive reaction in the basolateral plasma membrane of the normal acinar and ductal epithelia. The hyperplastic ductal epithelium in tumour specimens generally showed an increased staining for CA IX. Of 29 malignant tumours of exocrine pancreas, 10 showed moderate or strong immunoreaction for CA IX. The signal for CA XII remained weak in most malignant lesions. The present results show that both CA IX and XII are unevenly expressed in the ductal and acinar compartments of the human pancreas. The expression of these isoenzymes in a relatively low number of malignant tumour specimens suggests that they have a limited value in diagnostic evaluation of pancreatic carcinoma. However, the increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis.

The growing carbonic anhydrase (CA) gene family includes 11 enzymatically active isozymes in mammals. Each of them has a characteristic cellular and subcellular distribution pattern. In this report, we demonstrate for the first time a nuclear protein with CA activity. A polypeptide recognized by CA II antibodies was purified from several rat tissues using CA inhibitor affinity chromatography. This polypeptide of apparent 66 kDa mass was characterized using amino acid sequencing and CA activity measurements. It appeared to be identical to nonO/p54(nrb), a previously cloned and characterized RNA and DNA binding nuclear factor. Recombinant nonO generated in baculovirus bound to the CA inhibitor affinity chromatography matrix and revealed detectable CA activity (25 units/mg). Hansson's histochemical staining of rat lymph nodes followed by light and electron microscopy showed nuclear CA activity in lymphocytes, suggesting that the nuclear nonO protein is catalytically active in vivo. These results demonstrate that a previously known transcription factor is a novel, nonclassical CA. Through its CA activity, the nonO may function in the maintenance of pH homeostasis in the nucleus.

Background and Objective: Most patients with hereditary hemochromatosis are homozygous for a Cys282AETyr mutation in the HFE gene. This mutation has been shown to impair the association of the HFE gene product with b(2)-microglobulin and to prevent its cell surface presentation in transfected COS-7 and 293 cells. This study was performed to examine the expression of HFE protein in epithelial cells, macrophages, and circulating leukocytes obtained from normal subjects and patients with hereditary hemochromatosis., Design and Methods: Antisera against two different peptides of the HFE protein were used to immunostain tissue sections and isolate granulocytes, lymphocytes and monocytes., Results: Immunocytochemical staining showed that the HFE protein is expressed in gastric epithelial cells, tissue macrophages, and circulating monocytes and granulocytes. The cell surface associated signal, which was seen in normal gastric epithelial cells, monocytes and macrophages, was also present in C282Y mutant cells from patients with hereditary hemochromatosis, although at apparently reduced amounts in these cells., Interpretation and Conclusions: From these studies, it is clear that the C282Y mutation reduces but does not completely prevent presentation of the HFE protein on the cell surface of human monocytes, tissue macrophages, and gastric epithelial cells.

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 microM 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.

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.

The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues and biological fluids of the human body by catalysing the reversible reaction CO2 + H2O HCO3- + H+ (Davenport & Fisher, 1938; Davenport, 1939; Maren, 1967). Carbonic anhydrase isoenzyme VI (CA VI) is the only secretory isoenzyme of the mammalian CA gene family. It is exclusively expressed in the serous acinar cells of the parotid and submandibular glands, from where it is secreted into the saliva. In this review, we will discuss recent advances in research focused on the physiological role of salivary CA VI in the oral cavity and upper alimentary canal.

Carbonic anhydrases maintain pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O <=> HCO3- + H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands. Although it represents about 3% of the total protein in stimulated parotid saliva, its exact physiological significance in the saliva has not been established. In the present study, saliva samples were collected under strictly controlled conditions from young, healthy men and assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, alpha-amylase activity levels, lactobacillus and Streptococcus mutans counts were also determined, and the results were correlated with the dental status of the subjects. Salivary CA VI concentration, pH and buffering capacity values correlated negatively with the numbers of decayed, missing and filled teeth (DMFT index). The correlations between salivary CA VI concentration and DMFT index were most significant in subjects with poor oral hygiene. No correlation was found between salivary CA VI concentration and lactobacillus or Streptococcus mutans counts. As predicted, salivary lactobacillus and Streptococcus mutans counts showed a close positive correlation with the DMFT index. In contrast, no significant correlation was seen between salivary secretion rate or amylase activity and the DMFT index. The present results indicate that low salivary CA VI concentrations are associated with increased caries prevalence, particularly in subjects with neglected oral hygiene.

Salivary carbonic anhydrase (CA VI) appears to protect teeth from caries via mechanisms other than direct regulation of salivary pH and buffering capacity. To elucidate whether CA VI acts in the local microenvironment of the tooth surface, we studied the location and activity of the enzyme in the human enamel pellicle. The study was performed using a specific rabbit antiserum to human CA VI in conjunction with immunostaining and immunoblot techniques. CA activity was demonstrated using a histochemical staining method. CA VI immunostaining of extracted teeth having in vivo formed pellicle showed that the enzyme is present in the enamel pellicle. Immunostaining for salivary alpha-amylase, which is known to be present in the pellicle, showed a similar staining pattern. The presence of CA VI in the enamel pellicle was confirmed by immunoblotting of in vivo formed pellicle proteins. In vitro studies showed that CA VI binds to polished enamel surfaces from both saliva and solutions of purified enzyme. The intensity of the CA VI immunostaining on the enamel surface was dependent on the concentration of the applied enzyme. The histochemical staining of in vitro formed enamel pellicle confirmed that the bound enzyme retains its enzymatic activity. The presence of active CA VI in the human enamel pellicle suggests that it may accelerate the removal of acid by functioning locally in the pellicle layer on dental surfaces.

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)

Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets.

Carbonic anhydrase isoenzyme IX, MN/CA IX, is a recently discovered member of the carbonic anhydrase (CA) gene family with a suggested function in acid-base balance, intercellular communication, and cell proliferation. Increased expression of MN/CA IX has been observed with certain epithelial tumors. We investigated the expression of MN/CA IX in 69 colorectal neoplasms, consisting of 1 juvenile polyp, 8 hyperplastic polyps, 39 adenomatous lesions, 21 carcinomas, and 7 metastases. Tissue sections were immunostained with a monoclonal antibody specific to MN/CA IX. The proliferative activity of the tumor cells was evaluated by Ki-67 antigen immunoreactivity. The hyperplastic polyps showed a weak or moderate reaction for MN/CA IX only in the cryptal epithelium, as did the normal intestinal mucosa. The adenomas showed immunoreactivity mainly in the superficial part of the mucosa, whereas the distribution in the carcinomas and metastases was more diffuse. Comparative immunostaining of serial sections for Ki-67, a well established marker of cell proliferation, confirmed that MN/CA IX is expressed in areas with high proliferative capacity. Our results show abnormal MN/CA IX expression in colorectal neoplasms, suggesting its involvement in their pathogenesis. The co-occurrence of MN/CA IX and Ki-67 in the same tumor cells indicates its potential for use as a marker of increased proliferation in the colorectal mucosa.

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 C-terminal 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 fluorescence 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.

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.

Carbonic anhydrase VI (CA VI) is a secretory isoenzyme that, by analogy to alpha-amylase, is produced in the salivary glands and delivered into saliva. To determine whether CA VI is transferred into the circulation and is detectable in human serum, we collected blood samples from four healthy subjects at 3-h intervals throughout a 24-h period and measured concentrations of CA VI by a specific time-resolved immunofluorometric assay. All serum samples contained CA VI, the concentrations being approximately 22 times lower in serum than in the corresponding saliva samples. The presence of CA VI in serum was confirmed by Western blotting, which under reducing conditions identified a 42-kDa polypeptide band corresponding to the monomeric CA VI. The described time-resolved immunofluorometric assay for CA VI might be useful to identify or exclude diseases of the salivary glands in the differential diagnosis of patients whose serum amylase concentrations are increased.

Two successive saliva samples were collected under strictly standardized conditions from 209 healthy, selected male soldiers prior to and after breakfast in the morning and were assayed for carbonic anhydrase (CA) VI concentrations using a specific time-resolved immunofluorometric assay. Salivary secretion rates, pH and buffer capacity pH values, and amylase activity levels were also determined. CA VI concentrations correlated positively to salivary secretion rates and amylase activity levels. By contrast, no significant correlation was seen between CA VI concentrations and pH or buffer capacity pH values, nor between amylase activity levels and salivary secretion rates, pH or buffer capacity pH values. The smokers had unaltered salivary secretion rates, CA VI and amylase activity levels, but lower salivary pH and buffer capacity pH values than the non-smokers. Present results suggest that salivary CA VI is not directly involved in the regulation of pH in saliva.

Carbonic anhydrase (CA) II is the predominant CA isoenzyme in the brain of mammals. We have recently developed a dual-label time-resolved immunofluorometric assay to quantify minute amounts of CA I and II in the cerebrospinal fluid (CSF). The present study was aimed at elucidating the clinical value of such measurements in the case of neurological disorders. Lumbar CSF samples were obtained from 111 patients suffering from various neurological diseases and from 97 control patients with no specific signs of central nervous system diseases. The highest CA II concentrations were found in patients with brain infarction (median 66.5 micrograms L-1, n = 20), whereas the control patients had markedly lower values (median 7.8 micrograms L-1, n = 97). Relative to a reference range calculated from the control material (10.2 +/- 17.2 micrograms L-1), the sensitivity of CA II measurement in differentiating brain infarction was 100%. Patients with transient ischaemic attack (median 11.2 micrograms L-1, n = 9), multiple sclerosis (median 14.7 micrograms L-1, n = 18) or epilepsy (median 20.3 micrograms L-1, n = 17) usually had CA II concentrations within the normal range, but those with central nervous system infection (n = 14), dementia (n = 19) or trigeminal neuralgia (n = 6) tended to have higher CA II levels in their CSF, the median values being 39.1 micrograms L-1, 45.5 micrograms L-1 and 44.0 micrograms L-1 respectively. The findings indicate that the concentration of CA II in the CSF marks disease activity in patients with brain damage. This finding could provide a basis for further studies estimating the value of CA II measurement as a new laboratory marker of diseases affecting the brain.

Saliva contains several factors that protect the alimentary canal mucosa against acidity. We measured the secretory carbonic anhydrase (CA VI) levels in the saliva of patients with gastrointestinal disorders using a time-resolved immunofluorometric assay. The mean enzyme concentrations were found to be lower in patients with verified esophagitis, gastric ulcer, or duodenal ulcer than in control patients with nonacid peptic diseases. The biochemical data from the enzyme activity assays and western blots of the human gastric mucosa and gastric juice samples indicated that the swallowed CA VI probably retains its activity in the harsh environment of the gastric lumen. In the upper alimentary canal, CA VI may neutralize the acid by catalyzing the formation of carbon dioxide and water. The present findings suggest that drugs supplemented with CA VI may prove beneficial in treating acid-peptic diseases.

Alkaline hepatic bile is acidified in the gallbladder to prevent calcium precipitation and gallstone formation. Because membrane-bound carbonic anhydrase (CA) isoenzyme IV participates with cytoplasmic CA II in the acidification of urine in the kidney, we studied its expression in different regions of the human biliary tract using immunohistochemical techniques. The enzyme was expressed in the apical plasma membrane of the gallbladder epithelial cells and in the endothelium of the subepithelial capillaries. In the liver, some epithelial cells of the large bile ducts showed positive staining. Its presence in the gallbladder epithelium could be confirmed by Western blotting, which showed a single 35-kd polypeptide band, corresponding in molecular weight to the intact enzyme. The majority of the enzyme was phased to Triton X-114 detergent phase. A small amount of 35-kd polypeptide was also seen in the water phase. Smaller proteolytic fragments of the enzyme were not seen, suggesting that the tissue sample was well preserved. The results show that CA IV is expressed in abundance in the human gallbladder epithelium, where it may participate together with cytoplasmic CA II and ion transporters in acidification of the gallbladder bile via bicarbonate reabsorption.

We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.