Antibodies reacting to carbonic anhydrase isozymes (I and II) and albumin in sera from dogs.

IgGs to carbonic anhydrase isozymes (CA-I and CA-II) and albumin were identified in dog serum. IgG titers were determined in the sera of asymptomatic dogs, and in dogs with atopic dermatitis, diarrhea and/or vomiting, diabetes and/or pancreatitis, kidney disease, hepatic disease, and thyroid gland disease, using ELISA. Low titres of IgG-reactive CA-I, CA-II, BSA, and CSA were found in the sera of healthy beagles. Compared with healthy beagles, there was a significant difference in the titers of antibodies against CA-I in asymptomatic dogs, dogs with diabetes and/or pancreatitis, or thyroid gland disease, or hepatic disease. Compared with healthy beagles, there was a significant difference in the antibody titer of anti-CA-II IgG in asymptomatic dogs and in those with hepatic disease. There was a significant difference in the antibody titer of anti-BSA IgG between healthy beagles and dogs with hepatic disease.

Urinary carbonic anhydrase VI as a biomarker for kidney disease in pigs.

This study investigated whether carbonic anhydrase (CA)-VI has utility as a biomarker in swine kidney disease. Serum chemistry, histopathology, immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) analyses were performed. In the kidney of normal healthy pigs, CA-VI was localized in the epithelial cells of the renal distal straight tubules. CA-VI levels were 16 ± 35 ng/g wet tissue and 50 ± 66 ng/mL in normal pig kidney and urine, respectively, and 136 ± 173 ng/mL in the urine of pigs with kidney disease. CA-VI urinary concentration was not correlated with urinary urea nitrogen (UUN), urinary creatinine (Cre), or urinary albumin levels in pigs with kidney disease. However, UUN and Cre levels were positively correlated in the urine of pigs with kidney disease. These data suggest that urinary CA-VI may represent a biomarker for kidney disease in pigs, particularly for disorders affecting distal straight tubules.

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney.

BACKGROUND: Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear. RESULTS: CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys.A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues. The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous ß-actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887). CONCLUSIONS: The localisation of CA-VI indicates that it may play a specialised role in the kidney.

Measurement of Carbonic Anhydrase I and II Isozymes in Feces as a Marker of Occult Blood in Horses with Intestinal Tract Bleeding.

Although endoscopy is the definitive diagnostic method for the detection of colonic ulcers, the equipment required for performing the test is costly and difficult to use. Therefore, a simple cost-effective and reliable screening test for intestinal tract bleeding is needed. To this end, we measured carbonic anhydrase isozymes (CA-I and CA-II) originating from erythrocytes by ELISA in order to determine if they could be used as markers of occult blood in feces. For fecal extract preparation, 2 g of feces were mixed with 4 ml of 0.01 M Tris-HCl (pH 8.0) containing 0.01% thimerosal. The concentrations of CA-I and CA-II in the fecal samples of 13 clinically normal racehorses were found to be 30.0 ± 10.0 and 34.0 ± 13.0 ng/ml, respectively. Increased concentrations of CA-I were detected in the fecal samples of 5 horses after blood administration; however, no increase was observed in CA-II. The concentrations of CA-I and CA-II in the fecal samples of 88 racehorses with clinical signs of equine gastric ulcer syndrome (EGUS) were 115.3 ± 79.0 and 41.0 ± 42.0 ng/ml, respectively. Thus, our results indicate that CA isozymes can be useful as markers of occult blood in the fecal samples of horses with intestinal tract bleeding.

Binding affinity of anti-xylitol antibodies to canine hepatic vessels.

Xylitol is used as a sugar substitute in food products. Dogs have been reported to experience lethal liver injury after accidental ingestion of xylitol. Because liver injury may be a serious consequence of canine immune-mediated reactions, antibodies produced against xylitol may attack the liver. Therefore, in the present study, we evaluated whether binding sites for xylitol antibodies are located at the liver or not. Anti-xylitol antibodies were generated by immunization of rabbits with a xylose-bovine serum albumin conjugate. Immunohistological examination showed that binding sites for the anti-xylitol antibodies were located in the hepatic arteries and the portal veins. Western blotting analyses by using a canine liver homogenate showed 4 protein bands with different molecular weights which reacted with anti-xylitol antibodies. Therefore, binding of anti-xylitol antibodies to the vessels may be the first step in an immune-mediated pathogenic response in xylitol toxicity. Further studies are necessary to determine the effects of anti-xylitol antibodies on the liver in the pathogenesis of xylitol toxicity.

Muscle carbonic anhydrase III levels in normal and muscular dystrophia afflicted chickens.

BACKGROUND: The levels and immunohistochemical localization of muscle carbonic anhydrase III (CA-III) in healthy chickens and in muscular dystrophia affected (DA) chickens show that the muscles of diseased animal undergo a progressive increase of enzyme activity. METHODS: An enzyme-linked immunoassay was used to assess the CA-III levels in the muscles and other tissues from eight normal White Leghorn chickens and in two chickens with muscular dystrophy. Immunohistochemical localization of the enzyme in the muscles of these animals was also determined. RESULTS: The levels of CA-III in the tensor fasciae latae and the superficial pectoral muscles of the DA chickens were higher than the level in normal chickens. The concentrations of CA-III in erythrocytes and plasma from diseased chickens were approximately 15-fold and 1.4-fold higher than in the normal chickens, respectively. In the superficial pectoral and the tensor fasciae latae muscles of diseased chickens, the numbers of strongly stained and weakly stained fibers were greater than that in the normal chickens. CONCLUSION: The levels of CA-III in the superficial pectoral muscle, the tensor fasciae latae muscle, plasma and erythrocytes from the chickens with muscular dystrophy were higher than found in normal chickens.

Purification of chicken carbonic anhydrase isozyme-III (CA-III) and its measurement in White Leghorn chickens.

BACKGROUND: The developmental profile of chicken carbonic anhydrase-III (CA-III) blood levels has not been previously determined or reported. We isolated CA-III from chicken muscle and investigated age-related changes in the levels of CA-III in blood. METHODS: CA-III was purified from chicken muscle. The levels of CA-III in plasma and erythrocytes from 278 female chickens (aged 1-93 weeks) and 68 male chickens (aged 3-59 weeks) were determined by ELISA. RESULTS: The mean level of CA-III in female chicken erythrocytes (1 week old) was 4.6 µg/g of Hb, and the CA-III level did not change until 16 weeks of age. The level then increased until 63 weeks of age (11.8 µg/g of Hb), decreased to 4.7 µg/g of Hb at 73 weeks of age, and increased again until 93 weeks of age (8.6 µg/g of Hb). The mean level of CA-III in erythrocytes from male chickens (3 weeks old) was 2.4 µg/g of Hb, and this level remained steady until 59 weeks of age. The mean plasma level of CA-III in 1-week-old female chickens was 60 ng/mL, and this level was increased at 3 weeks of age (141 ng/mL) and then remained steady until 80 weeks of age (122 ng/mL). The mean plasma level of CA-III in 3-week-old male chickens was 58 ng/mL, and this level remained steady until 59 weeks of age. CONCLUSION: We observed both developmental changes and sex differences in CA-III concentrations in White Leghorn (WL) chicken erythrocytes and plasma. Simple linear regression analysis showed a significant association between the erythrocyte CA-III level and egg-laying rate in WL-chickens 16-63 weeks of age (p < 0.01).

Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues.

Swine secretory carbonic anhydrase VI (CA-VI) was purified from swine saliva and an antibody to CA-VI was generated. A specific and sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of swine CA-VI. The assay can detect as little as 5 ng/mL of swine CA-VI. Typical standard curves were determined for a range of CA-VI solutions (7.8 to 500 ng/mL). The coefficients of variation for these solutions were less than 5%. When 500, 250 or 100 ng/mL of swine CA-VI was added to swine sera, the recoveries were 102.0%, 109.7% and 100.2%, respectively. The concentrations of CA-VI in the saliva (26.2 ± 30.4 µg/mL), sera (3.3 ± 4.9 ng/mL), bile (153.0 ± 114.0 ng/mL), seminal plasma (124.0 ± 39.0 ng/mL) and parotid gland (441.3 ± 90.0 µg/g wet tissue), submaxillary gland (88.1 ± 124.4 µg/g wet tissue), sublingual gland (58.6 ± 24.6 µg/g wet tissue) and gallbladder (2.4 ± 1.3 µg/1g wet tissue) were determined by ELISA. The concentration of CA-VI in colostrum was 163.3 ± 101.4 ng/mL and did not decrease within 10 days following parturition. An immunohistochemical reaction to anti-CA-VI antiserum was observed in the columnar epithelial cells lining the gallbladder. These data suggest that secretory CA-VI plays various roles in pH regulation and the maintenance of ion and fluid balance.

Biochemical and developmental characterization of carbonic anhydrase II from chicken erythrocytes.

BACKGROUND: Carbonic anhydrase (CA) of the chicken has attracted attention for a long time because it has an important role in the eggshell formation. The developmental profile of CA-II isozyme levels in chicken erythrocytes has not been determined or reported. Furthermore, the relations with CA-II in erythrocyte and egg production are not discussed. In the present study, we isolated CA-II from erythrocytes of chickens and determined age-related changes of CA-II levels in erythrocytes. METHODS: Chicken CA-II was purified by a combination of column chromatography. The levels of CA-II in the hemolysate of the chicken were determined using the ELISA system in blood samples from 279 female chickens, ages 1 to 93 weeks, 69 male chickens, ages 3 to 59 weeks and 52 weeks female Araucana-chickens. RESULTS: The mean concentration of CA-II in hemolysate from 1-week-old female was 50.8 ± 11.9 mg/g of Hb. The mean levels of CA-II in 25-week-old (188.1 ± 82.6 mg/g of Hb), 31-week-old (193.6 ± 69.7 mg/g of Hb) and 49-week-old (203.8 ± 123.5 mg/g of Hb) female-chickens showed the highest level of CA-II. The levels of CA-II in female WL-chickens significantly decreased at 63 week (139.0 ± 19.3 mg/g of Hb). The levels of CA-II in female WL-chicken did not change from week 63 until week 93.The mean level of CA-II in hemolysate of 3-week-old male WL-chickens was 78.3 ± 20.7 mg/g of Hb. The levels of CA-II in male WL-chickens did not show changes in the week 3 to week 59 timeframe. The mean level of CA-II in 53-week-old female Araucana-chickens was 23.4 ± 1.78 mg/g of Hb. These levels of CA-II were about 11% of those of 49-week-old female WL-chickens. Simple linear regression analysis showed significant associations between the level of CA-II and egg laying rate from 16 week-old at 63 week-old WL-chicken (p<0.01). CONCLUSIONS: Developmental changes and sexual differences of CA-II concentration in WL-chicken erythrocytes were observed. The concentration of CA-II in the erythrocyte of WL-chicken was much higher than that in Araucana-chicken (p<0.01).

Immunohistochemical localization and gene expression of carbonic anhydrase isoenzymes CA-II and CA-VI in canine lower airways and lung.

The immunohistolocalization and gene expression of carbonic anhydrase (CA) isoenzymes CA-II and CA-VI in the canine lower airways and lung were examined using specific canine CA-II and CA-VI antisera and the RT-PCR method. Laryngeal, tracheal and bronchial epithelia, serous acinar and bronchiolar secretory cells and pulmonary great alveolar cells showed immunopositive reactions to anti-CA-II and anti-CA-VI antisera. However, all mucous cells showed immunonegative reactions. The physiological roles of CA-II and CA-VI in the lower airways and lung may involve the maintenance of pH balance and the protection of mucosal surfaces against the acidic milieu.

Determination of full-length cDNA nucleotide sequence of equine carbonic anhydrase VI and its expression in various tissues.

A full-length cDNA clone of an equine carbonic anhydrase (CA)-VI was obtained from the equine parotid gland. The cDNA sequence was 1338 bp long and was predicted to encode a 319 amino acid polypeptide with a putative signal peptide of 18 amino acids. The deduced amino acid sequence of mature CA-VI showed the similarity of 70% to those of other mammalians reported. Westernblot analysis using anti-horse CA-VI peptide detected the single band in parotid gland, and the band reduced its size by treatment with N-glycosidase F. Additionally, CA-VI protein expression was confirmed in submandicular gland and weakly in liver. In contrast, RT-PCR analysis revealed signals in the digestive tract including duodenum, jejunum, ileum, cecum and colon as well as the salivary glands. In addition, certain signals were detected in testis, thyroid gland and liver, but not in nerve tissue, skeletal muscle, spleen or lymph node.

Pathomechanism of cellular infiltration in the perivascular region of several organs in SAMP1/Yit mouse.

We investigated the histological changes of extra-intestinal organs, such as the liver, kidney, lung and pancreas in SAMP1/Yit mice, a human Crohn's disease model, using immunohistochemical techniques. The perivascular cellular infiltration was detected around the small vessels after 30 weeks. These infiltrating cells consisted of many CD4-positive T-lymphocytes, and small numbers of CD8- positive T-lymphocytes and IgG-positive B-lymphocytes. MAdCAM-1 and VCAM-1 were detected in vascular endothelial cells in non-affected regions of 13 and 20 week-old, as well as in the affected regions showing perivascular cellular infiltration after 30 weeks. In addition, integrin alpha4beta7 was detected on these infiltrating cells in the perivascular regions after 30 week-old. LT-beta and IL-12, cytokines of the Th-1-type immune response, were not observed in these affected regions. However, IL-4, one of the cytokines of the Th-2-type immune response, was detected on the perivascular infiltrating cells after 30 week-old. These results revealed that the changes in extra-intestinal organs were mainly caused by infiltration of CD4-positive T-lymphocytes into the perivascular regions in SAMP1/Yit mice. These cellular infiltrations were thought to be initiated by adhesion of CD4-positive T-lymphocytes to the endothelial cells mediated by MAdCAM-1 and integrin beta7. Immunohistochemistry for Th related cytokines indicated that the perivascular cellular infiltration was developed by the Th-2-type immune response in the extra-intestinal organs of SAMP1/Yit mouse.

Immunohistolocalization and gene expression of secretory carbonic anhydrase isoenzyme CA-VI in canine nasal cavity.

Immunolocalization of the secretory form of carbonic anhydrase isoenzyme, CA-VI were studied using a specific canine CA-VI antiserum, and CA-VI mRNA signals were also investigated using the reverse-transcriptase polymerase chain reaction (RT-PCR) in canine nasal mucosal epithelia and glands. Immunoreactivity to CA-VI was positive throughout the mucosal epithelial cells and in the cytoplasm of serous acinar and ductal epithelial cells of the nasal mucosa and glands, including the vestibule of the nose, but the mucous acinar cells of the glands were immunonegative. We detected CA-VI gene transcripts in the same regions as the CA-VI immunoreactivity. The physiological roles of CA-VI in the nasal mucosal epithelium and glands might maintain bicarbonate levels in nasal secretions and protect the mucosa against acid.

Immunohistochemistry of the bovine secretory carbonic anhydrase isozyme (CA-VI) in bovine alimentary canal and major salivary glands.

In the present study, we firstly demonstrated immunohistochemical expressions of secretory carbonic anhydrase (CA-VI) isozyme in bovine forestomach, large intestine and major salivary glands. CA-VI was detected in basal layer epithelial cells of esophageal and forestomach stratified epithelium, in mucous cells of upper glandular region of large intestine, in serous acinar cells of the parotid gland, in serous demilune cells and some ductal liner cells of mandibular, monostomatic sublingual and esophageal glands. These immunohistolocalizations suggested that bovine CA-VI plays various roles in pH regulation, maintenance of ion and fluid balance, and cell proliferation.

Immunohistolocalization of carbonic anhydrase isoenzymes (CA-I, -II and -III ) in canine epididymis.

The immunolocalization of the efferent duct and the epididymis in canine was firstly examined using an the immunohistochemical method with the canine carbonic anhydrase (CA) -I, CA-II and CA-III antisera. The efferent duct was immunonegative for all present canine CA antisera. However, some slender shaped epithelial cells in the head and body segments of the epididymal duct were intensely reacted to the CA-II antiserum. These results suggested that the CA-II might be controlled in the luminal environment in the head and body segments of the canine epididymis by the proton and bicarbonate balance for the maintenance of the spermatozoal stability and movement.

Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses.

Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/- 0.13 mg/g hemoglobin (Hb), respectively. Mean concentrations of CA-I and CA-II in riding horses were 2.34 +/- 0.52 and 0.76 +/- 0.08 mg/g Hb, respectively. When the CA levels in racehorses and riding horses were compared, the CA-I level in riding horses was higher than that in racehorses (p=0.01). The CA-II level in racehorses was higher than that in riding horses (p=0.02). These data suggest that the levels of CA isozymes in erythrocytes of racehorses were influenced by chronic physical stress. The CA-I concentration in erythrocytes of 2-month-old horses was approximately 0.25 mg/g Hb. The CA-I level noticeably increased during the first year of life and approached normal adult levels by 2 years. The CA-II level decreased slightly with age, indicating different regulation of CA-I and CA-II expression during development.

Immunohistolocalization of carbonic anhydrase isozyme (CA-VI) in bovine mammary glands.

The localization of bovine carbonic anhydrase isozyme VI (CA-VI) was examined immunohistochemically in bovine mammary glands during early lactation period (after 2-3 days of postpartum) and dry period (at about 2 months preparturition in adults), and young calves (at 30 and 150 days after birth) using specific CA-VI antiserum. The immunoreaction for anti-CA-VI antiserum was very weak in the mammary glands in young (prepubescent) calves. In dry period, CA-VI was also weakly expressed in secretory epithelial (acinar) and ductal cells. In contrast, the reaction was intense in mammary gland cells in early lactation period. Dot blotting analysis indicated that anti-CA-VI reacted positively to beastings and mature saliva, but weakly or not at all to milk during the dry period or calf saliva, respectively. The intense expression of CA-VI in the mammary glands in early lactation period might compensate for low levels of secretion from functionally and structurally immature salivary glands in young calves.

Nucleotide sequence and expression of a cDNA encoding canine carbonic anhydrase VI (CA-VI).

A full-length cDNA clone of a canine carbonic anhydrase VI (CA-VI) was generated from the canine parotid gland by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with degenerate primers designed from conserved regions of the same locus in humans and bovines employing RACE (rapid amplification of cDNA ends) techniques. The cDNA sequence was 1351 base pairs (bp) long and was predicted to encode a 320-amino-acid polypeptide containing a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI showed the highest similarity of 74% to that of human CA-VI. RT-PCR analysis with primers specific to the canine CA-VI demonstrated strong signals in the major salivary glands and weak signals in the minor salivary glands and esophagus of a healthy dog. No CA-VI mRNA was detected in the pancreas, liver or the digestive tract except the esophagus.

Immunohistochemical localization of carbonic anhydrase isozymes in the rat carotid body.

The rat carotid body was immunohistochemically stained for carbonic anhydrase I, II and III (CA-I, CA-II and CA-III). Immunoreactivity for CA-I was distributed in type I cells, type II cells and nerve bundles. Smooth muscle cells and endothelial cells of blood vessels were also strongly stained for CA-I. CA-II immunoreactivity was distinctly positive in type I cells and nerve bundles. Vascular smooth muscle cells were weakly positive, and type II cells were negative for CA-II. CA-III immunoreactivity was identified in type I cells and vascular smooth muscle cells. Our results suggest that carbonic anhydrase isozymes in type I cells play an important role in chemoreception for hypercapnia. Immunoreactivities for CA-I and CA-II in the nerve fibres may participate in the synergic action of carotid sinus nerve between hypoxic and hypercapnic stimuli.

Expression and localization of carbonic anhydrase in bovine mammary gland and secretion in milk.

Little attention has been paid to carbonic anhydrase VI (CA VI), a secretory type isozyme, in the bovine mammary gland, although the gland is an important exocrine gland and CA VI is known to localize in exocrine glands such as salivary and lacrimal glands in various animal species. In the present study mRNA expression and protein localization of CA VI in isolated gland tissues and in cloned epithelial cells from the mammary gland of Holstein cows (Bos taurus) were observed by reverse transcript polymerase chain reaction and immunocytochemistry. Also, changes of CA VI concentrations in milk were measured for 2 months postpartum by an enzyme-linked immunosorbent assay. CA VI gene expression was detected in the gland tissues and epithelial cells, and CA VI protein was localized in the cytoplasm of the epithelial cells. Colostrum contained the highest concentration of CA VI protein (100 ng/ml), decreasing in an exponential manner (P<0.001). We conclude that bovine mammary epithelial cells synthesize and secrete CA VI in colostrum at higher concentration than in normal milk, implying its role to compensate for low CA VI secretion in neonatal calves.