Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.     

Immunohistochemical Study of Colorectal Tumors for Expression of a Novel Transmembrane Carbonic Anhydrase, MN/CA IX, with Potential Value as a Marker of Cell Proliferation

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.     

Hereditary hemochromatosis gene (HFE) mutations C282Y, H63D and S65C in patients with idiopathic dilated cardiomyopathy

BACKGROUND: Hereditary hemochromatosis (HH), a common autosomal recessive disease, leads to excessive iron accumulation in some organs, including the heart. It is therefore not surprising that cardiomyopathy is one of the most severe complications of HH. The HFE gene defects have been thought to contribute to idiopathic dilated cardiomyopathy (IDCM) in some patients, even though the results of genotype analyses have so far been contradictory. Hence we set out here to evaluate the prevalence and potential role of HFE mutations in patients with IDCM. METHODS: A total of 91 IDCM patients and 102 controls were subjected to HFE mutation analyses, in which C282Y, H63D and S65C mutations were determined for each patient. We also analyzed the impact of the C282Y and H63D mutations on the left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and New York Heart Association (NYHA) functional classes. RESULTS: The prevalences of heterozygosity for the C282Y, H63D and S65C mutations in the IDCM patients were 13.2%, 22.0% and 2.2%, respectively. LVEDD was significantly higher (P=0.037) in those with the C282Y mutation at the end of the follow-up period than in those with no mutation. CONCLUSIONS: Our data showed no significant deviations in C282Y, H63D and S65C mutation frequencies between the IDCM patients and controls, suggesting that these mutations do not increase the risk of IDCM. Heterozygosity for the C282Y mutation may nevertheless be a modifying factor contributing to LV dilatation and remodeling.     

The relationship of glucose tolerance to sleep disorders and daytime sleepiness

The purpose of the study was to find out if snoring, sleep apnea and daytime sleepiness are independent indices of obesity related to type two diabetes (T2D), and whether depression is independently associated with features of sleep apnea. A population-based cohort study was conducted among 593 subjects (245 men and 348 women) born in 1935 and living in Oulu in 1996-1998. Glucose status was determined with a standard 2h oral glucose tolerance test, and sleeping disorders were recorded on the Epworth sleepiness scale (ESS) and in a questionnaire of five questions about sleeping and snoring. Depression was measured by the Zung self-rated depression scale. Insulin sensitivity was measured by quantitative insulin sensitivity check index. Habitual snoring was more common in diabetic subjects than in subjects with impaired glucose regulation (IGR) or normal glucose tolerance (NGT). All sleep disorders associated with neck circumference, waist circumference and body mass index (BMI). There was also a relationship between impaired insulin sensitivity and habitual snoring in bivariate analysis. In multiple logistic regression analysis, depression associated independently with daytime sleepiness (OR 3.00, 95% CI 1.40-6.46). Type 2 diabetes (T2D) (OR 1.93, 95% CI 1.04-3.57) and smoking (OR 1.69, 95% CI 1.00-2.84) associated independently with habitual snoring. BMI (OR 1.20, 95% CI 1.09-1.34) and male gender (OR 2.61, 95% CI 1.05-6.72) associated independently with sleep apnea. In a multiple regression model, BMI, neck circumference and habitual snoring associated independently with T2D. Habitual snoring was associated with T2D and impaired insulin sensitivity. Daytime sleepiness seemed to be linked with depression but not with using sleep medication, IGR and T2D.     

Ten-year natural course of habitual snoring and restless legs syndrome in a population aged 61–63?years at the baseline

Purpose

We investigated the prevalence, natural course, and associated risk factors of habitual snoring (HS) and restless legs syndrome (RLS) over a period of 10?years among an aging population from their early sixties to their seventies.

Methods

A population-based follow-up study among all persons born in 1935 and living in the city of Oulu in northern Finland was conducted. In this study, we examined subjects who had participated in two subsequent surveys conducted in 1996?C1998 and 2007?C2008. The data were gathered by questionnaires, as well as by laboratory and clinical measurements.

Results

Altogether 457 (55%) of the 838 eligible subjects participated in both surveys. The prevalence of both RLS and HS decreased during the 10?years from 21% to 15% and from 26% to 19%, respectively. Half of those who snored in 1996?C1998 stopped snoring in 10?years time, and half of those who suffered from restless legs at least three times per week in 1996?C1998 suffered from this syndrome never or less than once a week in 2007?C2008. The 10-year incidence of new cases of both HS and RLS was 7%. Male gender predicted best the incidence of new HS in 10?years, while the Zung sum score as a marker of depressive symptoms and waist circumference predicted RLS.

Conclusions

Overall, the prevalence of both HS and RLS seems to diminish during aging. The causes behind this still remain unknown and should be investigated with more sophisticated methods.     

PRRT2-related disorders: further PKD and ICCA cases and review of the literature

Recent studies reported mutations in the gene encoding the proline-rich transmembrane protein 2 (PRRT2) to be causative for paroxysmal kinesigenic dyskinesia (PKD), PKD combined with infantile seizures (ICCA), and benign familial infantile seizures (BFIS). PRRT2 is a presynaptic protein which seems to play an important role in exocytosis and neurotransmitter release. PKD is the most common form of paroxysmal movement disorder characterized by recurrent brief involuntary hyperkinesias triggered by sudden movements. Here, we sequenced PRRT2 in 14 sporadic and 8 familial PKD and ICCA cases of Caucasian origin and identified three novel mutations (c.919C>T/p.Gln307*, c.388delG/p.Ala130Profs*46, c.884G>A/p.Arg295Gln) predicting two truncated proteins and one probably damaging point mutation. A review of all published cases is also included. PRRT2 mutations occur more frequently in familial forms of PRRT2-related syndromes (80–100 %) than in sporadic cases (33-46 %) suggesting further heterogeneity in the latter. PRRT2 mutations were rarely described in other forms of paroxysmal dyskinesias deviating from classical PKD, as we report here in one ICCA family without kinesigenic triggers. Mutations are exclusively found in two exons of the PRRT2 gene at a high rate across all syndromes and with one major mutation (c.649dupC) in a mutational hotspot of nine cytosines, which is responsible for 57 % of all cases in all phenotypes. We therefore propose that genetic analysis rapidly performed in early stages of the disease is highly cost-effective and can help to avoid further unnecessary diagnostic and therapeutic interventions.     

Carbonic anhydrase IX deposits are associated with increased ascending aortic dilatation

Objectives. Carbonic anhydrase IX (CA IX) expression is induced by local hypoxia. We studied whether CA IX deposits associate with ascending aortic dilatation. Design. Aortic wall histology, CA IX expression, presence of leukocytes, plasma cells, macrophages, endothelial cells, smooth muscle cells, cell proliferation, elastin and collagen were studied in histological specimens collected from 30 patients who underwent surgery for ascending aorta. The samples were grouped according to presence of CA IX deposits. Results. Twenty out of 30 patients had CA IX-positive deposits within the adventitia, whereas 10 specimens remained negative. Adventitial inflammation was increased in CA IX-positive samples as compared with CA IX-negative ones (p?<?0.01). The mean diameter of the ascending aorta at the sinotubular junction increased significantly in patients with CA IX-positive staining as compared with CA IX-negative cases (63?±?3 vs 53?±?2?mm, p?<?0.02). Receiver operating characteristic curve analysis confirmed the association of CA IX positivity with increased ascending aortic dilatation (AUC 0.766; S.E. 0.090; p?=?0.020; 95% C.I. 0.590–0.941). Conclusions. Positive CA IX staining in certain aortic specimens suggests that increased CA activity may contribute to ascending aortic dilatation.     

Covalent adducts of proteins with acetaldehyde in the liver as a result of acetaldehyde administration in drinking water

BACKGROUND/AIMS: Acetaldehyde, the first metabolic product of ethanol, has been suggested to be responsible for several adverse effects of ethanol through its ability to form covalent adducts with proteins and cellular constituents. It has recently been suggested that acetaldehyde derived from microbial ethanol oxidation in the gut could also contribute to the effects of ethanol in the liver. The present work aimed to examine whether modification of proteins by acetaldehyde occurs in rat liver as a result of acetaldehyde administration in drinking water. METHODS: Rats were fed with either 0.7% acetaldehyde (n=10) or water (n=10) for 11 weeks. At the end of the feeding period, liver specimens were processed for immunohistochemistry for protein adducts with acetaldehyde and for hepatic cell type-specific protein markers. RESULTS: Mild fatty change was found in the liver of the acetaldehyde-treated animals but not in the control animals. Immunohistochemical stainings for acetaldehyde adducts revealed intensive positive staining for acetaldehyde adducts in eight (80%) of the animals fed with acetaldehyde. The adducts were predominantly perivenular, although positive staining also occurred along the sinusoids and in the periportal area. Double immunofluorescence staining experiments revealed that hepatocytes were the primary targets of acetaldehyde adduct deposition, although stellate cells and Kupffer cells also showed weak positive reactions. CONCLUSIONS: The present data indicate that acetaldehyde-protein adducts are formed in the liver of animals following acetaldehyde administration in drinking water, which may contribute to the hepatotoxicity of extrahepatic acetaldehyde. These findings should be implicated in studies on the extrahepatic pathways of ethanol oxidation.