Pei-Wen Pan

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.

S100P is a Ca2+ binding protein overexpressed in a variety of cancers, and thus, has been considered a potential tumor biomarker. Very little has been studied about its normal expression and functions.

Carbonic anhydrases (CAs) are physiologically important enzymes which participate in many gastrointestinal processes such as acid and bicarbonate secretion and metabolic pathways including gluconeogenesis and ureagenesis. The genomic data suggests that there are thirteen enzymatically active members of the mammalian CA isozyme family. In the present study, we systematically examined the mRNA expression levels of all known CA isozymes by quantitative real-time PCR in eight tissues of the digestive system of male and female 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.