The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk.

In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Apoptosis and apoptosis-related proteins in human endometrium.

Apoptosis has been shown to be an important regulator of endometrium function. To study the regulation of apoptosis in the endometrium during the normal menstrual cycle, the expression of the apoptosis related proteins Bcl-2 and Bax and their correlation to serum estradiol and progesterone, as well as to a replication-related antigen Ki-67 were analyzed. Nine uterine tissue samples and thirty-nine endometrial biopsy specimens were studied. Apoptosis was studied quantitatively by Southern blot analysis of internucleosomal cleavage of genomic DNA, and qualitatively by using in situ 3'-end labeling of fragmented DNA, and Bcl-2, Bax and Ki-67 were detected and quantified immunohistochemically. Apoptotic cells were mostly detected in the glandular epithelium of late secretory and menstruating endometrium. Immunostaining of Ki-67 was detected predominantly in proliferative endometrium. The expression of Bcl-2 was high in proliferative endometrium and decreased in the secretory phase, being very low or absent in the mid- and late secretory and menstruatory phases. Similarly, Bax expression also decreased, but was still present throughout the secretory phase. In human endometrium, apoptosis occurs predominantly in the late secretory and menstrual phases, and is related to alterations in the expression of Bcl-2 and Bax. A decrease in the Bcl-2/Bax ratio in the early secretory phase precedes DNA fragmentation and may predispose the cells to apoptosis.

Nuclear NonO/p54(nrb) protein is a nonclassical carbonic anhydrase.

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.

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.

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.