Low-avidity antibodies to carbonic anhydrase-I and -II in autoimmune chronic pancreatitis. (1/32)

Antibodies (Abs) to carbonic anhydrase (isoforms CA-I and CA-II) have been considered pathogenic factors in the development of autoimmune pancreatitis. Besides, such autoAbs might accelerate the pancreatic damage in alcoholic chronic pancreatitis (CP). The aim of the present study was to evaluate the presence of serum Abs to CA-I and CA-II in CP and the relative affinity of these Abs. Serum anti-CA-I and -CA-II Abs were measured in 89 patients with CP (48 alcoholic and 41 nonalcoholic) by an ELISA technique. The prevalence of those autoAbs in CP was compared with other autoimmune diseases where they have also been found. The presence of other serological manifestations of autoimmunity, such as hypergammaglobulinemia or antinuclear Abs, was determined in CP patients as well. Elevated serum levels of both anti-CA-I (24%) and -CA-II (18%) Abs were observed in CP, although their prevalence was lower than in autoimmune diseases like rheumatoid arthritis (44 and 25%, respectively) or systemic lupus erythematosus (39% for anti-CA-I Abs). Furthermore, these Abs were of low average avidity. On the other hand, a significantly higher proportion of nonalcoholic CP had anti-CA-II Abs with respect to alcoholic CP (15.2 vs. 2.4%, p < 0.05). Anti-CA-I and -CA-II Abs might be helpful in the diagnosis of autoimmune CP, and the detection of the latter Abs seems to discard alcoholic etiology. Although it does not discard any pathogenic role in autoimmune CP, the low-avidity of anti-CA Abs argues against such idea.  (+info)

Natural protein engineering: a uniquely salt-tolerant, but not halophilic, alpha-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments. (2/32)

Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea. An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities. It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability. Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration. Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl. Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I. Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions. Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.  (+info)

Taste buds and nerve fibers in the rat larynx: an ultrastructural and immunohistochemical study. (3/32)

We investigated the rat laryngeal taste buds and their innervation by electron microscopy and immunohistochemical methods. Taste buds were densely arranged in the surface facing the laryngeal cavity of the epiglottis, the aryepiglottic fold, and the cuneiform process of the arytenoid cartilages. The cells of the buds were classified into types I, II, III, and basal cells, the ultrastucture of which was almost the same as that previously reported in lingual taste buds. The type III cells that had synaptic contacts with nerve fibers were considered to be sensory cells. Immunohistochemical analysis revealed thick calbindin D28k-immunoreactive fibers and thin varicose fibers immunoreactive for calcitonin gene-related peptide or substance P in and around the taste bud. Serotonin-immunoreactive cells were also observed here. The results revealed the innervation pattern of laryngeal taste buds to be the same as that in lingual taste buds. Carbonic anhydrase (CA) is known to catalyze the hydration of CO2 and dehydration of H2CO3, and seems to be essential in CO2 reception. Immunoreactivity for CAI was detected in slender cells and that for CAIII was observed in barrel-like cells in the laryngeal taste buds. The pH-sensitive inward rectifier K+ (Kir) channel in the cell membrane may be involved in CO2 reception as well. CAII-reactive cells were also reactive to Kir4.1, PGP 9.5 and serotonin. Our results indicated that CAII and Kir4.1 are located in type III cells of the laryngeal taste buds, and supported the idea that the buds may be involved in the recognition of CO2.  (+info)

Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses. (4/32)

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.  (+info)

Expression of a novel carbonic anhydrase, CA XIII, in normal and neoplastic colorectal mucosa. (5/32)

BACKGROUND: Carbonic anhydrase (CA) isozymes may have an important role in cancer development. Some isozymes control pH homeostasis in tumors that appears to modulate the behaviour of cancer cells. CA XIII is the newest member of the CA gene family. It is a cytosolic isozyme which is expressed in a number of normal tissues. The present study was designed to investigate CA XIII expression in prospectively collected colorectal tumor samples. METHODS: Both neoplastic and normal tissue specimens were obtained from the same patients. The analyses were performed using CA XIII-specific antibodies and an immunohistochemical staining method. For comparison, the tissue sections were immunostained for other cytosolic isozymes, CA I and II. RESULTS: The results indicated that the expression of CA XIII is down-regulated in tumor cells compared to the normal tissue. The lowest signal was detected in carcinoma samples. This pattern of expression was quite parallel for CA I and II. CONCLUSION: The down-regulation of cytosolic CA I, II and XIII in colorectal cancer may result from reduced levels of a common transcription factor or loss of closely linked CA1, CA2 and CA13 alleles on chromosome 8. Their possible role as tumor suppressors should be further evaluated.  (+info)

Polyvinylpyrrolidone 40 assists the refolding of bovine carbonic anhydrase B by accelerating the refolding of the first molten globule intermediate. (6/32)

Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I(1)) to the second one (I(2)) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I(1), resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I(1). These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I(1) and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I(1) does not block the aggregation pathway directly but decreases the energy barrier for I(1) to refold to I(2) and thus reduces the accumulation of I(1). These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.  (+info)

Ultrahigh resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors reveal the molecular basis of high affinity. (7/32)

The atomic-resolution crystal structures of human carbonic anhydrases I and II complexed with "two-prong" inhibitors are reported. Each inhibitor contains a benzenesulfonamide prong and a cupric iminodiacetate (IDA-Cu(2+)) prong separated by linkers of different lengths and compositions. The ionized NH(-) group of each benzenesulfonamide coordinates to the active site Zn(2+) ion; the IDA-Cu(2+) prong of the tightest-binding inhibitor, BR30, binds to H64 of CAII and H200 of CAI. This work provides the first evidence verifying the structural basis of nanomolar affinity measured for two-prong inhibitors targeting the carbonic anhydrases.  (+info)

Immunohistolocalization of carbonic anhydrase isoenzymes (CA-I, -II and -III ) in canine epididymis. (8/32)

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.  (+info)

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