Ultramicroanalysis of pH,P_{CO_2 } and carbonic anhydrase activity at calcifying sites in cartilage

The mechanism by which (HCO ₃ ^– ) is elevated in extracellular cartilage fluids (C_fl) of rat tibial growth plates was investigated. Inin vitro studies, the pH- curves in a synthetic lymph were not detectably altered by proteinpolysaccharides or by a cationic protein. Also, (HCO ₃ ^– ) in C_fl aspirated from isolated incubates of growth cartilage decreased rapidly as a function of time. Results of both of these experiments mitigated against a role for cartilage secretions as the cause of blood-C_fl (HCO ₃ ^– ) gradientsin vivo. Acetazolamide administered to ratsin vivo reduced the blood-C_fl (HCO ₃ ^– ) gradient to an undetectable level. This effect could not be attributed to systemic acidosis produced by acetazolamide since control rats with a similar degree of systemic acidosis resulting from NH₄Cl treatment, maintained a substantial blood-C_fl (HCO ₃ ^– ) gradient. The distribution of carbonic anhydrase activity in epiphyseal and metaphyseal tissues of similar rats was determined by microassay. Enzymatic activity was not detected in cartilage samples, but was found in significant amounts in adjacent structures.This carbonic anhydrase activity measured in adjacent structures was hypothesized to represent sites of HCO ₃ ^– secretion. The possible involvement in HCO ₃ ^– secretion of epiphyseal or metaphyseal capillaries and bone cells is discussed.

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Zusammenfassung Der mechanismus, durch welchen (HCO ₃ ^– ) in extrazellulären Knorpelflüssigkeiten (_fl) der Wachstumsplatten von Rattentibiae erhöht ist, wurde untersucht. Beiin vitro Versuchen mit einer synthetischen Lymphe waren die pH- Kurven weder durch Proteinpolysaccharide noch durch kationisches Protein nachweisbar verändert. In C_fl, welche aus isolierten Inkubaten von Wachstumsknorpel entnommen wurden, nahm (HCO ₃ ^– ) in Funktion der Zeit ebenfalls rasch ab. Die Resultate beider Experimente sprechen dagegen, daß die Knorpelsekretein vivo als Ursache der Blut-C_fl (HCO ₃ ^– )- Gradienten in Betracht kommen. Acetazolamid, das Ratten verabreicht wurde, erniedrigte den Blut-C_fl (HCO ₃ ^– )-Gradienten auf ein nicht mehr nachweisbares Niveau. Dieser Effekt konnte nicht einer durch Acetazolamid hervorgerufenen generalisierten Acidose zugeschrieben, werden, da Kontrollratten mit einem ähnlichen Grad von generalisierter Acidose, welche von einer NH₄Cl-Behandlung herrührte, einen ansehnlichen Blut-C_fl (HCO ₃ ^– )-Gradienten aufrechterhielten. Die Verteilung der Kohlensäureanhydrase-Aktivität in epiphysären und metaphysären Geweben von gleichartigen Ratten wurde durch Mikroanalyse bestimmt. Eine enzymatische Aktivität konnte in den Knorpelproben nicht nachgewiesen werden, wurde jedoch in signifikanten Mengen in den angrenzenden Geweben gefunden. Es wurde die Hypothese aufgestellt, daß die Stellen, wo diese Kohlensäureanhydrase-Aktivität in angrenzenden Geweben gemessen wurde, den Sekretionsstellen von HCO ₃ ^– entspricht. Die mögliche Beteiligung von epiphysären und metaphysären Capillargefäßen und von Knochenzellen an, der HCO ₃ ^– -Sekretion wird diskutiert.

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Résumé Le mécanisme de l'élévation du (HCO ₃ ^– ) dans les liquides extracellulaires du cartilage (C_fl) a été étudié au niveau, de métaphyses tibiales de Rat. Au cours d'étudesin vitro, les courbes pH- dans une lymphe synthétique ne sont pas modifiées de façon nette par des protéines-polysaccharides ou par une protéine cationique. (HCO ₃ ^– ) de C_fl, aspiré à partir de pièces métaphysaires, incubées isolément, décroit rapidement en fonction du temps. Les résultats de ces deux expériences semblent infirmer un rôle des sécrétions cartilagineuses comme cause de gradients sang— C_fl (HCO ₃ ^– ) in vivo. L'acétazolamide, administré à des ratsin vivo, réduit le gradient sang —C_fl (HCO ₃ ^– ) jusqu'à, un seuil non dosable. Cette action ne peut être attribuée à l'acidose généralisée, produite par l'acétazolamide, étant donné que les rats témoins, ayant une acidose généralisée similaire, provoquée par un traitement à NH₄Cl, présentent un gradient sang —C_fl (HCO ₃ ^– ) net. La répartition de l'activité en anhydrase carbonique dans les tissus épiphysaires et métaphysaires de rats identiques est déterminée par micro-analyse. L'activité enzymatique n'est pas détectée dans des échantillons cartilagineux, mais est retrouvée, de façon significative, dans les structures adjacentes.L'activité en anhydrase carbonique, mesurée dans les structures adjacentes, est considérée comme les lieux de sécrétion d'HCO ₃ ^– . Le rôle éventuel des capillaires épiphysaires et métaphysaires et des cellules osseuses dans la sécrétion d'HCO ₃ ^– , est envisagé.

     

Calcium incorporation in matrix vesicles isolated from chicken epiphyseal cartilage

Summary Matrix vesicles isolated from chicken epiphyseal cartilage displayed an uptake of Ca²⁺ which was linear with time and the amount of vesicle protein. The matrix vesicles stimulated the incorporation of Ca²⁺ even at very low Ca × P, suggesting that they could bind Ca²⁺ and/or increase the local Ca × P to the metastable level. This uptake was abolished by EDTA or heating, and partially inhibited by cysteine, to the same extent as the hydrolysis of ATP. There was also a certain uptake of Ca²⁺ without added phosphate, this being stimulated by ATP up to 3 mmol, but diminishing again with higher concentrations. The presence of ATP failed to stimulate the uptake of Ca²⁺ more than an equimolar amount of phosphate in the form of inorganic KH₂PO₄. Mg²⁺ activated the hydrolysis ofp-nitrophenylphosphate at pH 10.5, and both Mg²⁺ and Ca²⁺ the hydrolysis of ATP from pH 7 to 9.5. Paradoxically, the omission of Mg²⁺ stimulated the uptake of Ca²⁺ several-fold.     

Localization of prostaglandin synthetase in chicken epiphyseal cartilage

Summary Prostaglandin synthetase activity in highspeed particulate fractions of chick epiphyseal cartilage has been characterized with respect to cofactor requirements, pH optimum, buffer-ion effects, types of prostaglandins formed, and the distribution of prostaglandin synthetase activity in zones of the epiphyseal plate.Direct homogenization of cartilage was found to be more efficacious than releasing chondrocytes by enzymatic digestion for preparation of prostaglandin synthetase, a homogenization time of 4 min yielding maximal activity. The optimal incubation medium contained 50 mM Tris buffer (pH 7.5), 2.5 mM epinephrine, 1 M hemoglobin, 3.25 mM glutathione, 200 g/ml enzyme protein, and 5 M substrate. Glutathione was effective only if present during homogenization. Rates of PGE₂ biosynthesis were linear up to 15 min and then rapidly declined, indicative of self-deactivation. The low levels of PGF₂ formed, and their decrease after 20 min incubation, suggests the possible presence of degradative enzymes.Prostaglandin synthetase was inhibited by aspirin, indomethacin, and vitamin E, but not vitamin K₁. Cation concentrations in the physiological range had only modest effects on prostaglandin biosynthesis, and then only if present during tissue homogenization. In the presence of phosphate buffer, Ca²⁺ was somewhat inhibitory. Since in the absence of phosphate Ca²⁺ had no deleterious effect, it is probably that the inhibitory effect was caused by precipitation of calcium phosphate.Hypertrophic and calcified cartilage exhibited significantly higher prostaglandin synthetase activity than the proliferating and maturing zones. The increased synthesis of prostaglandins in the low layers of the growth plate may indicate a role of these factors in chondrocyte differentiation and/or calcification.     

Correlation of lysozyme activity with proteoglycan biosynthesis in epiphyseal cartilage

Summary Pig epiphyseal cartilage (proximal ulna epiphysis) previously incubated in vitro in the presence of sodium [³⁵S]sulfate or [³H]thymidine was either analyzed by autoradiography or separated into 9 morphologically defined consecutive layers and investigated for³⁵S-incorporation into the guanidinium chloride-extractable proteoglycans and for lysozyme activity.The lowest³⁵S incorporation and lysozyme activity were determined in the zone of resting cells, but there is a consecutive increase in the rate of proteoglycan synthesis and lysozyme activity toward the diaphyseal cartilage-bone junction, with the maximum at the lower columnar cell zone and a sharp reduction of both parameters at the hypertrophic zone. The maxima of³⁵S incorporation and [³H]thymidine incorporation do not coincide.The guanidinium chloride-soluble proteoglycans exhibit macromolecular polydispersity. Fractions excluded from as well as retarded by Sepharose 2B gel could be separated and were detected in all zones.The results indicate a correlation of proteoglycan biosynthesis and lysozyme activity in epiphyseal cartilage.     

Developmental changes in carbonic anhydrase II in the rat kidney

We examined the distribution and maturational changes of carbonic anhydrase I (CAI) and carbonic anhydrase II (CAII) in microdissected nephron segments of Sprague-Dawley rats. CAI and CAII proteins were measured by enzyme-linked immunosorbent assay. CAI was not detected in any nephron segment in 7-week-old rats. CAII was present in the collecting ducts, proximal tubules, and thick ascending limbs of loop of Henle in 7-week-old rats. CAII contents were significantly higher in the early proximal tubules (S1) than in second (S2) and late (S3) portions of the proximal tubules, while the contents in S1 were less than in cortical collecting ducts (CCD), outer stripe and inner stripes of the outer medullary collecting ducts (OMCDo and OMCDi). CAII content in each of S1, CCD, and OMCD of 1-week-old rats was only 14% or less of that of adults, but increased steeply during the 2nd and 3rd weeks of life, reaching almost 40% at 3 weeks of age and 97% at 7 weeks. Our results indicate that CAII is present throughout the entire nephron of the rat, and that CAII content in S1, CCD, and OMCD increases exponentially during the first 7 weeks of life. Our data suggest that the immature low levels of CAII may explain, at least in part, the limited capacity of urinary acidification during neonatal life. Further studies are necessary to establish the role of such changes in CAII content in acid-base homeostasis during neonatal life. Received December 6, 1996; received in revised form September 8, 1997; accepted September 19, 1997     

A comparison of the effects of inhibitors of carbonic anhydrase on osteoclastic bone resorption and purified carbonic anhydrase isozyme II

Summary We have assessed the effects of five sulfonamides with widely varying inhibitory activity for carbonic anhydrase (CA) in the bone slice assay using disaggregated rat osteoclasts (OCs), and in the Maren assay where the catalytic activity of purified CA isozyme II (CA II) was measured. There was an excellent correlation between the relative potencies of the compounds in the two assays: ethoxzolamide (ETH)>acetazolamide (AZ)>M&B 21659>M&B 9811>M&B 7973. In the bone slice assay, ETH and AZ were found to be the most potent inhibitors of OC bone resorption, with IC₅₀ values of 0.09 and 0.8 μM, respectively (from plan surface area of bone resorbed). These results support previous observations showing that OCs use CA II to generate protons during bone resorption and that CA II activity is essential for OCs to be able to resorb bone.     

Immunohistochemical and biochemical demonstration of calcium-dependent cysteine proteinase (calpain) in calcifying cartilage of rats

Calpain is a Ca2(+)-dependent cysteine proteinase that has neutral pH optima. There are two classes of calpains that differ in their optimal calcium ion concentration for enzymatic activity. Calpain I requires a low concentration of Ca2+ for activation, and calpain II requires a much higher Ca2+ concentration. This report describes the immunohistochemical and biochemical demonstration of calpain II in calcifying cartilage in rats and also the degradation of the cartilage proteoglycan subunit by calpain II. Immunoperoxidase (peroxidase-antiperoxidase) staining of the frozen sections of the knee joint from 3-day-old and 6-day-old Wistar rats, using polyclonal antibodies against the respective heavy subunits of calpains I and II, showed positive staining only with the anti-calpain II antibody in the hypertrophic chondrocytes and surrounding cartilaginous matrix of the growth cartilage. Diethylaminoethyl-cellulose chromatography of the cartilaginous extract from 3-day-old rats showed a peak of caseinolytic activity attributable to calpain as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anti-calpain II antibody of the calpain peak demonstrated identity with the heavy subunit of calpain II (80 kDa). Proteoglycan-degrading activity of calpain was assessed using porcine kidney calpain II and the porcine articular cartilage proteoglycan subunit. After incubation in the presence of Ca2+, degradation of proteoglycan was demonstrated by the change of the elution position on Sepharose-2B chromatography. It is possible that calpain functions as one of the proteoglycan-degrading proteolytic enzymes of growth cartilage. Intracellular localization of calpain in hypertrophic chondrocytes also suggests a role in the hypertrophic process of the chondrocyte in growth cartilage.     

Stimulation of carbonic anhydrase in osteoclasts by parathyroid hormone

Summary Changes in the acidity of osteoclasts were evaluated by direct measurement of the fluorescent intensity of osteoclasts exposed to acridine orange, a fluorescent weak base which becomes concentrated in acid-containing subcellular compartments. Parathyroid hormone (PTH) produced dose-dependent increases in fluorescent intensity; maximal increases in intensity occurred at doses between 3 and 10 μg PTH/ml of culture medium. Acetazolamide, a potent inhibitor of carbonic anhydrase, inhibited the increase in fluorescence induced by PTH, but this drug was less effective in reducing fluorescence in maximally than in submaximally stimulated osteoclasts, indicating that either more enzyme or more resistant enzyme was present in the PTH-stimulated cells. Because increased fluorescence of acridine orange is a sign of greater acidity, these results suggest that (1) PTH stimulates the acidity of osteoclasts, (2) carbonic anhydrase activity is necessary for maximum acidity, and (3) carbonic anhydrase is activated by PTH.     

Changes in leukocyte migration during carbonic anhydrase activity inhibition

Experiment 1 evaluated changes in leukocyte migration during acetazolamide (AZ) inhibition of carbonic anhydrase activity in leukocytes. AZ induced changes in the intracellular calcium concentration, and extracellular calcium is thought to be a factor inducing an increase in leukocyte migration. Next, Experiment 2 determined whether extracellular calcium concentration was a primary factor influencing leukocyte migration in the absence of AZ. The distance of leukocyte migration increased in a dose-dependent manner with AZ despite the presence of IL-8 or LPS in Experiment 1. The extracellular calcium concentration used in the present study had no influence on the distance in leukocyte migration in Experiment 2. The distance of leukocyte migration showed a tendency to increase in a dose-dependent manner with LPS concentration. In conclusion, AZ may stimulate leukocyte migration due to its participation in the regulation of intracellular pH controlled by CA activity without an effect of low extracellular calcium concentration. In addition, AZ was thus suggested to possibly have an anti-inflammatory effect in supporting leukocyte migration during inflammatory reactions.     

Animal model of chondrocyte apoptosis in the epiphyseal cartilage of the neonatal bone

Apoptosis is considered to be the mechanism responsible for the death of chondrocytes during endochondral bone formation. It is also claimed that apoptosis of the chondrocytes is age related and that the apoptotic index increases with age. However, a detailed analysis of the apoptotic activity of the neonatal epiphyseal cartilage is lacking. A model that evaluates apoptosis in the femoral rat epiphyseal cartilage both quantitatively and qualitatively is reported. Apoptotic incidence in the epiphyseal cartilage reached a maximum at age 6 days, but the age in our study did not significantly affect the percentile rate of apoptotic chondrocytes (P > 0.05, Kruskal-Wallis test). Apoptosis in the zone of hypertrophic cartilage played the most important role in the growth plates homeostasis. Morphologic evidence of apoptosis was necessary in addition to positive nick end labeling of cells. Electron microscopy studies revealed atypical modes of programmed death of the growth plate chondrocytes in addition to the classical apoptotic mode.An erratum to this article can be found at     

Electrolytes of isolated epiphyseal chondrocytes,matrix vesicles,and extracellular fluid

Summary Chondrocyte, matrix vesicle, and membrane fractions, as well as interstitial fluid samples from the proliferating and hypertrophic zones of chicken epiphyseal cartilage were analyzed for electrolyte content. Intracellular Ca levels were 1.4–2.1 mM, over 90% of which was nondiffusible. Isolated hypertrophic chondrocytes had higher intracellular Na and lower K than proliferating cells. Matrix vesicles contained 25 to 50 times higher concentrations of Ca than the adjacent cells. Vesicles from the zone of hypertrophy contained twice as much Ca as did those from the proliferating area. Ca/P₁ molar ratios of matrix vesicles were much higher than those of cells or of later mineral deposits. These findings indicate that Ca is concentrated in matrix vesicles during formation, but acuumulation of Ca and P₁ must continue in the matrix. X-ray diffraction of freeze-dried vesicle and membrane fractions failed to detect crystalline apatite, suggesting that crystals seen in electron micrographs of matrix vesicles may be artifacts. Interstitial fluid expressed from epiphyseal cartilage was higher in K, P_i, Mg and nucleotides, and lower in Na and Cl, than blood plasma. Fluid from the hypertrophic zone was higher in K and nucleotides, but not P_i or Mg, than that from the proliferating layer. These data suggest that selective leakage or extrusion of these constituents, which are normally intracellular, must occur, especially in the hypertrophic zone. More of the Ca and Mg, and less of the P_i, was protein-bound in cartilage fluid than in blood plasma. There was more binding of the divalent cations in fluid from proliferating than from hypertrophic cartilage. The presence of greater amounts of ultrafilterable peptides in fluid from hypertrophic than from proliferating cartilage or blood plasma, suggests that proteolytic activity may release bound divalent cations during mineralization.     

Vitamin E stimulates trabecular bone formation and alters epiphyseal cartilage morphometry

The effects of dietary vitamin E (VIT E) and lipids on tissue lipid peroxidation and fatty acid composition, epiphyseal growth plate cartilage development, and trabecular bone formation were evaluated in chicks. A 2×2 factorial design was followed using two levels (30 and 90 IU/kg of diet) of dl--tocopheryl acetate and two different dietary lipids. The basal semipurified diet contained one of the following lipid treatments: anhydrous butter oil (40 g/kg)+ soybean oil (60 g/kg), [BSO], or soybean oil (100 g/kg), [SBO]. After 14 days of feeding, the level of -tocopherol in plasma was higher and thiobarbituric acid reactive substances (TBARS) were less in plasma and liver of chicks supplemented with 90 IU of VIT E compared with those given 30 IU of VIT E. Body weights and tibiotarsal bone lengths were not affected by the dietary treatments Saturated fatty acids (14:0, 15:0, 16:0, 17:0, and 18:0) were increased in tibiotarsal bone of chicks fed the BSO diet. In contrast, total polyunsaturated fatty acids and the ratio of unsaturated fatty acids/saturated fatty acids were higher in plasma of chicks fed SBO compared with the values from chicks fed BSO. The thickness of the entire growth plate cartilage and the lower hypertrophic chondrocyte zone was significantly greater in chicks fed 90 IU/kg of VIT E. Kinetic parameters on bone histomorphometry indicated that mineral apposition rate was higher in chicks fed 90 IU/kg of VIT E. The interaction effect between the VIT E and BSO treatments led to the highest trabecular bone formation rate among the groups. These data suggest that VIT E protects against cellular lipid peroxidation in cartilage to sustain normal bone growth and modeling.Approved as Journal Paper Number 14556 of the Purdue Agricultural Experiment Station     

碳酸酐酶IX表达在肾透明细胞癌预后评估中的价值

目的 评估肾透明细胞癌组织中碳酸酐酶IX(CA IX)表达在患者预后判断中的价值.方法 应用免疫组织化学P-V方法检测 120例肾透明细胞癌和25例正常肾组织石蜡标本中CA IX的表达.以肿瘤特异性生存率作为最终和主要的评估目标.运用Cox回归模型行CA IX表达与预后关系的单因素和多因素分析,以P＜0.05为差异有统计学意义.结果 112例(93.3%)获随访,随访6～94个月,中位时间45个月,无瘤生存75例,带瘤生存3例,死亡34例,其中死于肿瘤28例.正常肾组织均不表达CA IX.120例肾透明细胞癌组织中CA IX高表达89例(74.2%),高表达者中获随访82例,无瘤生存62例(75.6%),带瘤生存2例(2.4%),死亡18例(22.0%),死于肿瘤13例(15.9%),复发和(或)转移9例(11.0%),中位生存期为92个月.肾透明细胞癌CA IX低表达31例(25.8%),其中获随访30例,无瘤生存13例(43.3%),带瘤生存1例(3.3%),死亡16例(53.3%),死于肿瘤15例(50.0%),中位生存期为53个月,复发和(或)转移8例(26.7%).2组肿瘤特异性生存率比较经log-rank检验,差异有统计学意义(P=0.000,χ2=15.950),CA IX高表达组1、3、5、7年肿瘤特异性生存率分别为95.2%、83.9%、81.2%、78.2%,CA IX低表达组分别为89.5%、63.9%、46.8%、40.1%.2组术后肿瘤复发和(或)转移率比较差异有统计学意义(P=0.040,χ2=4.200).多因素Cox 回归模型分析显示CA IX表达是影响肾透明细胞癌预后的指标(RR=0.186).结论 CA IX高表达与肾透明细胞癌患者术后死亡率及肿瘤复发和(或)转移率呈负相关,CA IX可作为判断肾透明细胞癌预后的指标.

Abstract：

Objective To evaluate the prognostic significance of carbonic anhydrase IX (CA IX) expression in patients with clear cell renal cell carcinoma (ccRCC). Methods CA IX expression in a cohort of 120 patients with ccRCC was evaluated by P-V immunohistochemistry with a rabbit CA IX polyclonal antibody. Twenty-five normal kidney tissues were used as a control. The relationship between CA IX expression and prognosis was analyzed by univariate and multiple-factor analysis (Cox regression model). The primary end point was cancer specific survival. Results One hundred and twelve (93.3%) patients were followed up with the median follow-up time of 45 months (range, 6 to 94 months). Seventy-five patients survived without evidence of tumor recurrence, 3 patients survived with tumor recurrence, and 34 patients died, 28 of the 34 died of cancer. CA IX expression was negative in all normal renal tissue. High CA IX expression was observed in 89 (74.2%) patients, among which 82 patients were followed up, and the disease free survival was 75.6% (62/82). Two (2.4%) patients survived with tumor recurrence, and 18 (22.0%) patients died, of which 13 (15.9%) died of cancer. Tumor recurrence and (or) metastasis occurred in 9 (11.0%) patients, with a median survival of 92 months in this high expression group. Low CA IX expression was observed in 31 (25.8%) patients, among which 30 patients were followed up, and the disease free survival was 43.3% (13/30). One (3.3%) patient survived with tumor recurrence, and 16 (53.3%) patients died, of which 15 (50.0%) died of cancer. Tumor recurrence and (or) metastasis occurred in 8 (26.7%) patients with a median survival of 53 months in this low expression group. Cancer specific survival between CA IX high expression group and low expression group was significantly different (P=0.000, χ2=15.950), and tumor relapse and (or) metastasis rates were significantly different (P=0.040, χ2=4.200). The 1, 3, 5 and 7 year cancer specific survival rates were 95.2%, 83.9%, 81.2% and 78.2% respectively in the high CA IX expression group, and 89.5%, 63.9%, 46.8% and 40.1% respectively in the low expression group. Multivariate analysis with Cox regression model showed that CA IX expression was a prognostic factor (RR=0.186). Conclusions High CA IX expression is negatively correlated with postoperative mortality, relapse and (or) metastasis in ccRCC. CA IX expression could be used as a prognostic biomarker in ccRCC.     

Effect of vitamin D status on the activity of carbonic anhydrase in chicken epiphysis and kidney

Summary Chickens were raised for 6 weeks from the date of hatch under red light on a vitamin D-free diet; controls were given an oral vitamin D supplement. Vitamin D-deficient animals showed decreased total serum calcium concentration and decreased DNA content in epiphysis and kidney homogenates. In calcifying epiphysis, total carbonic anhydrase (CA) activity was decreased, but activity per μg DNA was slightly increased and specific activity was double that of the controls. Polyacrylamide gel isoelectric focusing after preparation of the enzyme showed a picture similar to that seen after parathyroid hormone (PTH) administration in chicks; therefore, this could be considered a secondary hyperparathyroidism. The CA activation was not seen in the kidney which can be explained by induction of an endogenous inhibitor protein of the cyclic AMP-dependent protein kinase exclusively in the kidney in vitamin D deficiency. In an additional experiment, chickens were raised for 3 weeks from the date of hatch under red light on a vitamin D-free diet. Daily oral substitution by different vitamin D metabolites (1,25 (OH)₂D₃, 25OHD₃, 24, 25(OH)₂D₃) over 7 days led to CA activation compared with controls probably by restoring protein kinase activity in the kidney. Our results show that CA activity is inversely correlated with serum calcium concentrations which is in agreement with a regulatory mechanism recently proposed by us.     

大鼠胫骨骺板软骨细胞P物质的表达

目的 研究P物质（SP）在大鼠胫骨骺板软骨细胞中的表达及其在长骨纵向生长中的意义。方法 应用免疫组化技术检测8周龄大鼠胫骨骺板不同组织学层次软骨细胞的SP表达状况。结果 骺板静止及增殖期软骨细胞未见SP免疫阳性染色，肥大区及钙化区软骨细胞可见SP免疫组化阳性染色。结论 骺板肥大区及钙化区软骨细胞可以表达SP，提示SP在长骨的纵向生长中可能起调节作用。     

Aberrant expression of membranous carbonic anhydrase IX (CAIX) is associated with unfavorable disease course in papillary and clear cell renal cell carcinoma

Objective

Antibodies against carbonic anhydrase IX (CAIX) are often part of immunohistochemical panels used to assist renal cell cancer (RCC) subtyping. This study was undertaken to determine, whether assessing CAIX expression levels could provide additional prognostic information.

Localization of H+-ATPase and carbonic anhydrase II in ameloblasts at maturation

The localization of vacuolar-type H⁺-ATPase and carbonic anhydrase II (CA II) in rat incisor enamel organs at maturation was examined by light and electron microscopy. The immunoreactivity for both vacuolar-type H⁺-ATPase and CA II was intense on the ruffled border of ruffle-ended ameloblasts (RA), but moderate at the distal end of smooth-ended ameloblasts (SA). Immuno-gold particles indicated that CA II was not confined to the ruffled border of RA alone, but also distributed in the cytoplasm of RA and SA. These findings suggest that RA may secrete protons produced by CA II via the ruffled border into enamel by active transport of vacuolar-type H⁺-ATPase. Secreted protons may activate hydrolytic enzymes to degrade the organic components of enamel matrix. Vacuolar-type H⁺-ATPase on vesicles of SA suggests that a specific configuration of ruffled borders in RA may be formed by the fusion of vesicle membranes in the distal end of cytoplasm of SA.     

Absorption et redistribution du calcium dans le manteau des lamellibranches en relation avec la structure

The lamellibranch mantle has been identified as the tissue responsible for secreting the chemical constituents of shell. The relationship structure and function of the central part of the freshwater clam mantle was studied. Autoradiographic localization of the calcium pools of the mantle, using⁴⁵Ca, suggests that the ionized pool quickly exchangeable, is located mainly in the cytoplasm of the epithelial cells and calcium reserves occur as small extracellular granules localized in the interstitial tissue of the mantle. The kinetics of calcium movement and shell organic matrix formationin vivo are discussed in the light of the present observations.     

Soluble and resistant proteoglycans in epiphyseal plate cartilage

The proteoglycans of cartilage occur in a form which is readily extracted (soluble) and in form which is relatively difficult to extract (resistant). Following the extraction of the soluble proteoglycans from slices of epiphyses from young rats, the distribution of the resistant proteoglycans are visualized by staining with toluidine blue. Daily quantitative recoveries of uronic acid over 7 days are used as an index of the rate and completeness of extraction. In contrast to other cartilages (nasal, costal, ear, articular) in which the resistant proteoglycans are restricted to perilacunar localizations, the resistant proteoglycans in epiphyseal plate extend across the plate as a continuous stratum and occupy extraterritorial regions. This stratum of resistant proteoglycans is difficult to identify with a specific zone in the plates of young individuals, because of primitive columniation. In more highly organized, older human and porcine epiphyseal plates, however, the stratum is clearly seen at the junction of the zones of resting and proliferating chondrocytes. It dips down a short distance between the columns, disappears and then reappears again at the level of the zone of provisional calcification. These observations are discussed in the context of endochondral growth.

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Zusammenfassung Die Knorpelproteoglycane kommen in einer leicht extrahierbaren (löslichen) und in einer relativ schwer extrahierbaren (resistenten) Form vor. Nach der Extraktion der löslichen Proteoglycane aus Epiphysenschnitten junger Ratten wird die Verteilung der resistenten Proteoglycane durch Toluidinblau-Färbung aufgezeigt. Als Index für die Geschwindigkeit und Vollständigkeit der Extraktion wird die tägliche quantitative Ausbeute von Uronsäure während 7 Tagen verwendet. Im Gegensatz zu anderen Knorpelarten (Nasen-, Rippen-, Ohren- und Gelenkknorpel), bei welchen die resistenten Proteoglycane nur perilacunär vorkommen, gehen die resistenten Proteoglycane der Epiphysenplatte über die Platte als zusammenhängende Schicht hinaus und treten in extraterritorialen Bereichen auf. Diese Schicht resistenter Proteoglycane kann in den Platten junger Individuen wegen der ursprünglichen Säulenbildung nur schwierig als eine bestimmte Zone identifiziert werden. In höher organisierten, älteren Epiphysenplatten des Menschen und des Schweines ist die Schicht jedoch deutlich an der Berührungsstelle der Zonen ruhender und proliferierender Chondrocyten ersichtlich. Sie setzt sich eine kurze Strecke zwischen den Säulen fort, verschwindet dann aber und erscheint wieder auf der Höhe der vorläufigen Verkalkungszone. Diese Beobachtungen werden mit dem endochondralen Wachstum in Zusammenhang gebracht.

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Résumé Les protéoglycanes du cartilage se présentent sous une forme que l'on peut extraire facilement (soluble) et sous une formule difficile à extraire (résistante). Après extraction de protéoglycanes de coupes d'épiphyses de jeunes rats, le répartition des protéoglycanes résistantes est visualisée par coloration au bleu de toluidine. La détermination quantitative quotidienne d' acide uronique pendant 7 jours est utilisée comme indice de la vitesse et de l'efficacité de l'extraction. Contrairement à d'autres cartilages (nasal, costal, oreille, articulaire) où les protéoglycanes résistantes sont limitées à des régions périlacunaires, les protéoglycanes résistantes de la métaphyse s'étendent au-delà sous forme d'une couche continue et occupent des régions extra-territoriales. Cette couche de protéoglycanes résistantes est difficile d'identifier avec une zone spécifique dans la métaphyse de jeunes individus, par suite d'un alignement primitif. Cependant au niveau de métaphyses humaines ou de porcs plus âgés, cette couche est nettement visible à la jonction des zones de chondrocytes au repos et en division. Elle s'étend sur une courte distance entre les cellules sériées, disparait et réapparait à nouveau au niveau de la zone de calcification temporaire. Ces résultats sont discutés en fonction de la croissance enchondrale.

     

Matrix vesicles in chicken epiphyseal cartilage. Separation from lysosomes and the distribution of inorganic pyrophosphatase activity

Summary The extracellular matrix vesicles from epiphyseal cartilage of chickens were isolated by differential centrifugation. The matrix vesicles obtained showed considerable activity of lysosomal enzymes. This appears to have been due to lysosomal contamination because when we used a new density gradient medium (Percoll?), the lysosomal enzyme activities and the activity of alkaline phosphatase could be totally separated. Electron microscopy of the alkaline phosphatase-rich fraction showed matrix vesicle-like structures. Phosphatase activities of the cells and matrix vesicles were further studied by Sephadex G-200 gel filtration. Specific magnesium-activated inorganic pyrophosphatase, distinct from nonspecific alkaline phosphatase, could be demonstrated in the cellular fraction. No such separate activity could be demonstrated in the matrix vesicle fraction, and it is supposed that the pyrophosphatase activity in the matrix vesicles originates from the alkaline phosphatase.