Perivascular cells in cartilage canals of the developing mouse epiphysis

Morphological variability among perivascular cells adjacent to cartilage matrix during the elongation of canals through both uncalcified and calcified matrix has not been reported. Cartilage canals were located in distal femoral epiphyses of 5- to 7-day-old mice and identified as vascular channels arising from perichondrial surfaces along the condyles and intercondylar fossae. Three stages of canal development were identified based on the length of canals and on characteristics of chondrocytes and matrix surrounding the canals. Superficial canals terminated in uncalcified matrix of resting cartilage; intermediate canals terminated in matrix containing hypertrophic chondrocytes; deep canals terminated in calcified matrix. The ultrastructural morphology of perivascular cells in contact with the matrix varied in the three stages. Cells resembling fibroblasts and vacuolated macrophages were present adjacent to the uncalcified matrix in superficial canals. At the tips of intermediate canals, cells resembling fibroblasts were larger, contained numerous lysosomes and phagolysosomes, and were in intimate contact with the matrix. At the tips of deep canals, chondroclasts with ruffled borders and clear zones contacted the calcified matrix. The results indicate that (1) mouse epiphyses provide a suitable model for studying cartilage-canal perivascular cells, (2) calcification of cartilage matrix occurs along the course of the canal, and (3) the morphology of perivascular cells in contact with the matrix may be determined, in part, by matrix calcification.     

Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells

M cells in Peyer's patch follicle epithelium endocytose and transport luminal materials to intraepithelial lymphocytes. We examined (1) enzymatic characteristics of the epithelium covering mouse and rat Peyer's patches by using cytochemical techniques, (2) distribution of lectin-binding sites by peroxidase-labeled lectins, and (3) anionic site distribution by using cationized ferritin to develop a profile of M cell surface properties. Alkaline phosphatase activity resulted in deposits of dense reaction product over follicle surfaces but was markedly reduced over M cells, unlike esterase which formed equivalent or greater product over M cells. Concanavalin A, ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin reacted equally with M cells and with surrounding enterocytes over follicle surfaces. Cationized ferritin distributed in a random fashion along microvillus membranes of both M cells and enterocytes, indicating equivalent anionic site distribution. Staining for alkaline phosphatase activity provides a new approach for distinguishing M cells from enterocytes at the light microscopic level. Identical binding of lectins indicates that M cells and enterocytes share common glycoconjugates even though molecular groupings may differ. Lectin binding and anionic charge similarities of M cells and enterocytes may facilitate antigen sampling by M cells of particles and compounds that adhere to intestinal surfaces in non-Peyer's patch areas.     

颅骨锁骨发育不良患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的水平：1例报告

背景：骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶与骨骼的发育密切相关。 目的：观察颅骨锁骨发育不良患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的变化。 方法：收集1例颅骨锁骨发育不良患者临床资料,利用全颌曲面断层片检查全面了解患者的颌骨发育情况;详细进行口腔检查,记录恒牙萌出及乳牙迟萌以及埋伏牙、多生牙等;综合临床资料和实验室检查确定临床诊断,并检测患者骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶的变化。 结果与结论：该患者有锁骨发育不良、囟门未闭合以及多生牙、埋伏牙等颅骨锁骨发育不良的典型症状,可诊断为颅骨锁骨发育不良,但患者染色体无异常。实验室检查发现患者的骨特异性碱性磷酸酶和抗酒石酸酸性磷酸酶均增高,提示颅骨锁骨发育不良患者可能伴有抗酒石酸酸性磷酸酶基因的变异。     

Cytochemical and biochemical demonstration of an alkaline phosphatase in human red cells

Summary Activity of alkaline phosphatase has been demonstrated in the red cells of three members of a family with erythrocytosis. The highest activity is found in the cytoplasm of erythroblasts in bone marrow.K _m value and migration velocity of the extracted erythrocyte enzyme in gels differ from that of leukocyte alkaline phosphatase. At last part of the enzyme is bound to red cell membrane or stroma.

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Zusammenfassung In den Erythrocyten und Erythroblasten von drei Mitgliedern einer Familie mit Polyglobulie wurde alkalische Phosphatase nachgewiesen. Die stärkste Aktivität fand sich in den Erythroblasten des Knochenmarkes.K _m -Wert und elektrophoretische Wanderungsgeschwindigkeit des extrahierten Erythrocytenenzyms in Gelen unterscheiden sich von den Werten der alkalischen Neutrophilenphosphatase. Zumindest ein Teil des Enzyms ist an die Erythrocytenmembran oder das Erythrocytenstroma gebunden.

     

Immunohistochemistry of collagen types II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible

We investigated the immunohistochemical localisation of types II and X collagen as well as the cytochemical localisation of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible on d 14–16 of pregnancy. On d 14 of pregnancy, although no immunostaining for types II and X collagen was observed, alkaline phosphatase activity was detected in all cells in the anlage of the future condylar process. On d 15 of pregnancy, immunostaining for both collagen types was simultaneously detected in the primarily formed condylar cartilage. Alkaline phosphatase activity was also detected in chondrocytes at this stage. By d 16 of pregnancy, the hypertrophic cell zone rapidly increased in size. These findings strongly support a periosteal origin for the condylar cartilage of the fetal mouse mandible, and show that progenitor cells for condylar cartilage rapidly or directly differentiate into hypertrophic chondrocytes.     

Widespread expression of tartrate-resistant acid phosphatase (Acp 5) in the mouse embryo

Tartrate-resistant acid phosphatase (TRAP, Acp 5) is considered to be a marker of the osteoclast and studies using 'knockout' mice have demonstrated that TRAP is critical for normal development of the skeleton. To investigate the distribution of TRAP in the mammalian embryo, cryostat sections of 18 d murine fetuses were examined by in situ hybridisation, immunohistochemistry and histochemical reactions in situ. Abundant expression of TRAP mRNA was observed in the skin and epithelial surfaces of the tongue, oropharynx and gastrointestinal tract including the colon, as well as the thymus, ossifying skeleton and dental papillae. TRAP protein was identified at the same sites, but the level of expression in the different tissues did not always correlate with apparent enzyme activity. The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.     

Cytochemical localization of acid phosphatase and adenosine triphosphatase in some avian mechanoreceptors.

Ultrastructural distribution of acid phosphatase and adenosine triphosphatase was studied in the receptor elements of HERBST and GRANDRY sensory corpuscles. Acid phosphatase activity was established in the elements of smooth and rough endoplasmic reticulum of perineural capsule cells, as well as in the secondary lysosomes of all cell types. Particular interest was paid on the activity of myelin-like dense bodies and some clear core vesicles belonging to the axoplasm of receptor nerve fibres. Adenosine triphosphatase activity was established on the membranes of receptor structures and pinocytotic vesicles. More deposits of electron dense material were localized on the axolemma of the non-myelinated portions of the receptor nerve fibres. The functional significance and importance of the both enzymes in the receptor structures was discussed.     

Structure, formation and role of cartilage canals in the developing bone

In the long bones, endochondral bone formation proceeds via the development of a diaphyseal primary ossification centre (POC) and an epiphyseal secondary ossification centre (SOC). The growth plate, the essential structure for longitudinal bone growth, is located between these two sites of ossification. Basically, endochondral bone development depends upon neovascularization, and the early generation of vascularized cartilage canals is an initial event, clearly preceding the formation of the SOC. These canals form a discrete network within the cartilaginous epiphysis giving rise to the formation of the marrow space followed by the establishment of the SOC. These processes require excavation of the provisional cartilaginous matrix which is eventually replaced by permanent bone matrix. In this review, we discuss the formation of the cartilage canals and the importance of their cells in the ossification process. Special attention is paid to the enzymes required in disintegration of the cartilaginous matrix which, in turn, will allow for the invasion of new vessels. Furthermore, we show that the mesenchymal cells of the cartilage canals express bone-relevant proteins and transform into osteocytes. We conclude that the canals are essential for normal epiphyseal bone development, the establishment of the growth plate and ultimately longitudinal growth of the bones.     

Ultrastructural localization of alkaline phosphatase activity in the developing thyroid gland of the rat

The localization of alkaline phosphatase activity during morphogenesis of the thyroid gland was studied at the fine structural level in Holtzman rats from the fifteenth day of foetal life till the first day after birth. The present work deals with the formation of thyroid follicles in the median thyroid primordium only. Since the rat thyroid does not develop synchronously, the three stages described may overlap during a given day of development. In the precolloid stage that extends roughly from the fifteenth to the nineteenth day of development, alkaline phosphatase activity is localized in Golgi saccules and vesicles and also in smooth membrane tubules and vesicles found in the cytoplasm near the lateral plasma membranes. At the end of this stage the lateral plasma membranes become strongly reactive and a cluster of positive vesicles and tubules appears immediately under the junctional zone. The second phase, the early colloid stage (17–19 days), is characterized by the formation of the colloid cavity in center of the disc-like junctional zone: at this moment the newly formed apical plasma membrane bearing the microvilli shows the reaction product. During the third stage (18 days onwards), which starts with a gradual increase in the diameter of follicular lumina, a drastic fall in alkaline phosphatase activity is observed. In one day old rats, follicular cells are completely negative. These findings are briefly discussed in connection with thyroid cell differentiation.     

Ultrastructural localization of Tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteocalsts, and chondrocalsts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and α-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(β,γ-methylene) diphosphate, and β-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl₂ and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.     

Nuclear localization of alkaline phosphatase in cultured human cancer cells

The nucleolar localization of alkaline phosphatase (ALPase) was observed by electron microscopic cytochemistry. The culture cells used in this study were normal human cells (fibroblast, WI-38) and human cancer cells (hepatocellular carcinoma, Hep-G2; malignant melanoma, A-375; pancreatic carcinoma, BxPC-3). Cultured cells in almost all strains contained high ALPase activity in the nucleolus, and the localization of ALPase changed during the cell cycle stages. The pattern of ALPase localization during the interphase was divided into three groups: cytoplasmic type, nucleus type, and both types. Moreover, at the mitotic phase, the reaction products were observed on the chromosome. In the cultured malignant melanoma cells, the appearance ratio of ALPase reaction products on the nucleolus (33.9%) showed a higher ratio compared with normal cultured fibroblasts (6.3%). This phenomenon suggests that the high level of the ALPase reaction product may be related to the high level of proliferation of cancer cells.     

Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.     

Basement membrane composition of cartilage canals during development and ossification of the epiphysis

Background: Cartilage canals are perichondral invaginations of blood vessels and connective tissue that are found within the epiphyses of most mammalian long bones. Functionally, they provide a means of transport of nutrients to the hyaline cartilage, a mechanism for removal of metabolic wastes, and a conduit for stem cells that are capable of initiating and sustaining ossification of the chondroepiphysis. Morphological and biomolecular changes of the chondroepiphyses appear to potentiate vascular invasion and enable regional formation of secondary centers of ossification within the chondroepiphyses of developing bones. Methods: As both cell migration and vascular invasion are anchorage dependent processes, antibodies to laminin and Type IV collagen were used to assess compositional changes in the basement membrane of cartilage canals accompanying epiphyseal ossification. Results: Differences in chronological appearance, as well as, in distribution between the two components were noted in the chondroepiphysis. Laminin was distributed throughout the connective tissue of cartilage canal at all stages of developement, and not limited to an association with the vascular lumen. Type IV collagen was not Present during the initial perichondral invagination. Although staining for Type IV collagen was later acquired, its distribution was restricted to a discontinuous rimming of the periphery of the canal, and a diffuse presence within the intra-canalicular mesenchyme. Conclusions: Concurrent with chondrocyte hypertrophy and mineralization of the hyaline matrix, rapid changes in both the morphology of the vessel and distribution of the antibodies were detected. In addition to the presence of laminin at the interface of the endothelium and the hyaline matrix, a wide distribution within the connective tissue components of the newly ossifying matrix of epiphyseal bone could be detected. Type IV collagen remained closely associated with the lumens of the intra-canalicular vessels throughout the transition. Following ossification of the secondary center, staining for Type IV collagen could then be detected in the boneforming regions of transforming matrix as well, clearly delineating the individual vessels within the newly formed marrow spaces. This suggests that bone formation is intimately related to vessel staining for collagen type IV, and that acquired vessel competence is a facet of endochondral bone formation that results from provisional matrix changes. Furthermore, the data suggests that during bone formation under tension, basement membrane deposition can be demonstrated without an intermediary hyaline matrix hypertrophic chondrocyte phase. This data was interpreted to suggest that chondrocyte hypertrophy at the growth plate may be a reaction to vascular invasion, that in turn, stimulates adjacent chondrocyte proliferation. © 1995 Wiley-Liss, Inc.     

Comparison of the distribution patterns of tenascin and alkaline phosphatase in developing teeth,cartilage, and bone of rats and mice

Tenascin is a glycoprotein of the extracellular matrix, which has been associated with differentiation of hard tissue forming cells. Alkaline phosphatase (AP) is involved in calcification, and it has also been suggested to function in cell differentiation. We have compared the distributions of tenascin and AP in the developing skull and teeth of embryonic and growing rats and mice. Tenascin was localized by immuno-Peroxidase and AP by enzyme histochemical staining of tissue sections. Both tenascin and AP were largely restricted to bone, cartilage, and teeth. In cartilage, tenascin was expressed in the perichondrium, whereas AP activity was detected only in the hypertrophic cartilage. In growing intramembranous bone, tenascin and AP were expressed in the periosteum and endosteum. AP activity was restricted to the inner layer of the periosteum, whereas tenascin expression extended to the more superficial layers. In bud-staged teeth tenascin but no AP activity was localized in the condensing mesenchymal cells around the epithelial bud. At the bell stage both tenascin and AP activity were localized in the cuspal mesenchyme, and the intensity of staining decreased towards the cervical region. In summary, tenascin was present at all sites of AP activity except in the epithelial cells of the enamel organ and the hypertrophic cartilage of the mandibular condyle. In mesenchymal tissues tenascin was more widely distributed than AP. It can be suggested that tenascin has functions at earlier stages of hard tissue formation than AP.     

Classification of normal and malignant lymphatic cells using acid phosphatase and acid esterase

Summary The usefulness of cytochemical tests (APh and ANAE) to replace or to supplement membrane markers in subclassification of normal and malignant lymphatic cells was investigated. Material: normal lymphocytes subfractionated by rosetting and centrifugation, and in M. Hodgkin and CLL; lymphoblastoid cell lines; malignant lymphatic cells in different types of lymphatic leukemias. In normal human blood, T-lymphocytes are marked by a distinct dot-like ANAE-reactivity which is somewhat less pronounced in the small (11%) subgroup of Fc-IgG-receptor positive T-lymphocytes; B-lymphocytes are negative or finely granular positive. Lymphoblastoid cell lines of B- and of T-type are ANAE- and APh-positive. In some lymphatic malignancies, a characteristic pattern of activity of APh or of ANAE may support the diagnosis. The value of ANAE-cytochemistry is highly estimated for the quantitative determination of the percentage of normal T-lymphocytes in lymphatic leukemias, immunological disorders, and during immunosuppressive therapy.

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Abbreviations ANAE acid -naphthyl acetate esterase - APh acid phosphatase - (c-)ALL (common-type) acute lymphatic leukemia - CLL chronic lymphatic leukemia - E(AET)-lymphocytes lymphocytes spontaneously forming rosettes with (AET-treated) SRBC - Fc-IgG-lymphocytes lymphocytes forming rosettes with IgG-sensitized ORBC - MGG May-Grünwald-Giemsa staining of blood films - Non-E-lymphocytes lymphocytes not forming rosettes with SRBC (from interphase of Ficoll-separation) - ORBC ox red blood cells - SRBC sheep red blood cells - (T)-ALL (T-cell-type) acute lymphatic leukemia - T-IgG-lymphocytes E(AET)-lymphocytes forming rosettes with IgG-sensitized ORBC - T-non-IgG-lymphocytes E(AET)-lymphocytes non-rosetting with IgG-sensitized ORBC