Presence Of Alkaline Phosphatase Activity Research Articles

Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

Differential strain response in alkaline phosphatase activity to available phosphorus in Microcoleus autumnalis

Toxic, benthic cyanobacterial proliferations have increased in frequency and severity globally and can have negative impacts on aquatic ecosystems, recreation and human health. Microcoleus autumnalis has been associated with numerous animal fatalities and is causing increasing concern. It tends to grow in systems with moderate dissolved inorganic nitrogen and very low dissolved reactive phosphorus. Acquisition of nutrients, particularly phosphorus, from organic sources may explain how M. autumnalis can reach the high biomass in these relatively nutrient deplete environments. In the present study the effect of phosphorus concentration and source on alkaline phosphatase activity was investigated in toxic and non-toxic M. autumnalis strains. Toxic strains exhibited significantly higher alkaline phosphatase activity than non-toxic strains (p < 0.05), and alkaline phosphatase activity increased in all strains under phosphorus-depleted conditions (p < 0.05). Alkaline phosphatase activity was also present in environmental M. autumnalis mats, though at lower levels than in laboratory experiments. The presence of alkaline phosphatase activity indicates that the acquisition of phosphorus from organic phosphorus sources may contribute to the ability of M. autumnalis to grow in systems with low dissolved reactive phosphorus.

Osteogenic potency of human bone marrow mesenchymal stem cells from femoral atrophic non-union fracture site

Objective: Mesenchymal stem cells (MSCs) exist in the site of atrophic non-union fracture. The aim of this study was to evaluate the osteogenic potency of MSCs in order to have a better understanding of the unclear pathophysiology of atrophic non-union fracture Methods: This is an in vitro experimental study. Sample was obtained from the non-union site of a patient with a 6-years-history of atrophic non-union fracture of right femur. The MSCs was isolated from the fracture site and was cultured in the growth medium. Confirmation of the MSCs was performed and then osteogenic differentiation was performed in mono-layered MSC grown in both home-made and commercial osteogenic media. To evaluate the osteogenic differentiation, we performed Alizarin red staining and colorimetric assay for alkaline phosphatase (ALP). Results: From Alizarin red staining, most cells in the osteoblast medium were stained red by the staining. The result of colorimetric assessment of ALP shows that peak concentration was reached after 4 minutes in osteogenic group and control group. Conclusion: The presence of ALP activity and positive Alizarin red staining in our study showed that MSCs stem cells obtained from site of atrophic non-union is capable to be differentiated into osteogenic cells. . J Clin Exp Invest 2014; 5 (2): 159-163

Development of a New Approach to Aid in Visual Identification of Murine iPS Colonies Using a Fuzzy Logic Decision Support System

The a priori identification of induced pluripotent stem cells remains a challenge. Being able to quickly identify the most embryonic stem cell-similar induced pluripotent stem cells when validating results could help to reduce costs and save time. In this context, tools based on non-classic logic can be useful in creating aid-systems based on visual criteria. True colonies when viewed at 100x magnification have been found to have the following 3 characteristics: a high degree of border delineation, a more uniform texture, and the absence of a cracked texture. These visual criteria were used for fuzzy logic modeling. We investigated the possibility of predicting the presence of alkaline phosphatase activity, typical of true induced pluripotent stem cell colonies, after 25 individuals, with varying degrees of experience in working with murine iPS cells, categorized the images of 136 colonies based on visual criteria. Intriguingly, the performance evaluation by area under the ROC curve (16 individuals with satisfactory performance), Spearman correlation (all statistically significant), and Cohen's Kappa agreement analysis (all statistically significant) demonstrates that the discriminatory capacity of different evaluators are similar, even those who have never cultivated cells. Thus, we report on a new system to facilitate visual identification of murine- induced pluripotent stem cell colonies that can be useful for staff training and opens the possibility of exploring visual characteristics of induced pluripotent stem cell colonies with their functional peculiarities. The fuzzy model has been integrated as a web-based tool named “2see-iPS” which is freely accessed at http://genetica.incor.usp.br/2seeips/.

Effects of transplanted mesenchymal stem cells isolated from Wharton’s jelly of caprine umbilical cord on cutaneous wound healing; histopathological evaluation

The aim of this study was to investigate the effects of transplanted Wharton's jelly mesenchymal stem cells (WJMSCs) of caprine umbilical cord on cutaneous wound healing process in goat. After collection of caprine pregnant uterus of mixed breed goats from abattoir, the Wharton's jelly (WJ) of umbilical cord was harvested. The tissues were minced in ventilated flasks and explant culture method was used for separating mesenchymal stem cells (MSCs). The isolated cells were immunostained for Actin protein, histochemically assayed for the presence of alkaline phosphatase activity, and analyzed for detection of matrix receptors (CD44) and hematopoetic lineage markers (CD34), using flow cytometery. After The isolated cells, 3×10(6) MSCs were stained with BrdU and prepared for transplantation to each wound. Four 3-cm linear full thickness skin incisions were made on both sides of thoracic vertebrate of four Raeini goats (two wounds on each side). The left wounds were implanted with MSCs in 0.6 ml of Phosphate buffer saline (PBS), and the right wounds considered as control group that received 0.6 ml of PBS. The samples were taken from the wounds 7 and 12 days after the wounding, and healing process was compared histologically between the two groups. Anti-BrdU staining showed that the transplanted cells were still alive in the wound bed during the study. The histopathological study revealed that re-epithelialization was complete at days 7 in treated wounds with WJMSCs, whereas in control wound the wounds still showed incomplete epithelialization 12 days after wounding. Also, microscopic evaluation showed less inflammation, thinner granulation tissue formation with minimum scar in the treated wounds in comparison with control wounds. In conclusion, this study demonstrates the beneficial effect of caprine WJMSCs in cutaneous wound healing in goat.

Hypertrophic differentiation and calcification during intervertebral disc degeneration

In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.

A simple approach for mouse embryonic stem cells isolation and differentiation inducing embryoid body formation

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.

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Enzyme‐labeled fluorescence (ELF) and bulk alkaline phosphatase (AP) activity enzyme assays were used to evaluate the phosphorus (P) status of phytoplankton communities in San Francisco and Monterey bays. Both regions exhibit spatial and temporal variability in bulk AP activity with maximum activities during the early spring and summer periods of high biological productivity. ELF analysis revealed pronounced differences in the makeup of organisms responsible for AP activity in these two environments. In Monterey Bay dinoflagellates are responsible for the bulk of the AP activity. Diatoms infrequently exhibited AP activity. Dinoflagellates that comprised only 14% of all cells counted in Monterey Bay accounted for 78% of AP‐producing cells examined. The presence of AP activity in this group suggests that changes in P sources, concentrations, and bioavailability could disproportionably influence this group relative to diatoms in Monterey Bay. In San Francisco Bay, AP production, indicated by ELF, was associated primarily with bacteria attached to suspended particles, potentially used to hydrolyze organic compounds for carbon, rather than to satisfy P requirements. Our results highlight the importance of organic P as a bioavailable nutrient source in marine ecosystems and as a component of the marine P cycle.

Alkaline phosphatase activity in the phytoplankton communities of Monterey Bay and San Francisco Bay

Enzyme-labeled fluorescence (ELF) and bulk alkaline phosphatase (AP) activity enzyme assays were used to evaluate the phosphorus (P) status of phytoplankton communities in San Francisco and Monterey bays. Both regions exhibit spatial and temporal variability in bulk AP activity with maximum activities during the early spring and summer periods of high biological productivity. ELF analysis revealed pronounced differences in the makeup of organisms responsible for AP activity in these two environments. In Monterey Bay dinoflagellates are responsible for the bulk of the AP activity. Diatoms infrequently exhibited AP activity. Dinoflagellates that comprised only 14% of all cells counted in Monterey Bay accounted for 78% of AP-producing cells examined. The presence of AP activity in this group suggests that changes in P sources, concentrations, and bioavailability could disproportionably influence this group relative to diatoms in Monterey Bay. In San Francisco Bay, AP production, indicated by ELF, was associated primarily with bacteria attached to suspended particles, potentially used to hydrolyze organic compounds for carbon, rather than to satisfy P requirements. Our results highlight the importance of organic P as a bioavailable nutrient source in marine ecosystems and as a component of the marine P cycle.

146 LECTIN DOLICHOS BIFLOURUS AGGLUTININ (DBA) IS A SPECIFIC MARKER for PRIMITIVE GERM CELLS IN NEONATAL PORCINE TESTIS

Livestock transgenesis is primarily directed towards improving productive traits or for biomedical research. Transgenic pig is of immense interest because it could provide tissues and organs for xenotransplantation to humans. Conventional methods like pronuclear microinjection of zygote and, more recently, somatic cell nuclear transfer (SCNT) have been used for generating genetically modified pigs. These methods are, however, inefficient and have numerous shortcomings. Male germline stem cells have enormous potential, because they could serve as an alternative approach to generate genetically modified pigs, in which the homologous recombination technique could be applied. However, this could not be achieved due to lack of knowledge of culture conditions and markers that can distinguish germ cells from somatic cells, enabling their isolation and identification in culture. Lectin dolichos biflourus agglutinin (DBA), which has specific affinity for ?-d-N-acetyl-galactosamine, reacts with the large round primordial germ cells of the pig genital ridge (Takagi et al. 1997 Mol. Reprod. Dev. 46, 567–580). Immunohistochemical analysis of testicular samples from different age groups revealed that DBA binds to primitive germ cells in neonatal pig testis like gonocytes and pre-spermatogonia. Variable DBA affinity among germ cells and its progressive loss with age suggested that DBA bound strongly to gonocytes, weakly to undifferentiated spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase activity in germ cells from neonatal pig testis further confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were isolated using 2-step enzymatic digestion and discontinuous Percoll density gradient. Approximately 70% gonocytes were present in the fraction 5 (50–60% gradient) that were identified by their DBA affinity. Isolated cells were cultured in DMEM/F12 supplemented with 10 �g mL-1 of insulin, 10 �g mL-1 of apo-transferrin, 100 U mL-1 of penicillin, 50 �g mL-1 of streptomycin, 40 �g mL-1 of gentamycin sulphate, 1 mM pyruvate, 1� non-essential amino acid solution, and 10% fetal bovine serum at 37�C in 5% CO2 in air in a humidified atmosphere. Initially, gonocytes grew as adherent cells that formed foci of flat colonies. These colonies grew in size during the culture period, and DBA-positive cells were observed. Flat colonies transformed to ES-like colonies by 6–7 days of culture, which were still positive for DBA binding. Proliferation of germ cells was evaluated by double immunostaining with DBA and anti-bromodeoxyuridine (BrdU) antibody following BrdU incorporation, suggesting that 53–55% DBA-positive cells were in S phase after 7 days of culture. In conclusion, lectin DBA is a marker for porcine primitive germ cells in neonatal pig testis including gonocytes that could be used for their identification during isolation and in culture. Isolated gonocytes could proliferate in vitro without specific growth factor supplementation.

Growth/differentiation factor 5 enhances chondrocyte maturation.

Growth/differentiation factor 5 (GDF5) is required for limb mesenchymal cell condensation and joint formation during skeletogenesis. Here, we use a model consisting of long-term, high-density cultures of chick embryonic limb mesenchymal cells, which undergo the entire life history of chondrocyte development, to examine the effects of GDF5 overexpression on chondrocyte maturation. Exposure to GDF5 significantly enhanced chondrocyte hypertrophy and maturation, as determined by the presence of alkaline phosphatase activity, collagen type X protein production, and the presence of a sulfated proteoglycan-rich extracellular matrix. Histologic analysis also revealed an increase in cell volume and cellular encasement in larger lacunae in GDF5-treated cultures. Taken together, these results support a role for GDF5 in influencing chondrocyte maturation and the induction of hypertrophy in the late stages of embryonic cartilage development, and provide additional mechanistic insights into the role of GDF5 in skeletal development.

Alkaline phosphatase activity in bovine oocytes and preimplantation embryos as affected by removal of the zona pellucida and culture medium constituents.

The aims of the present study were: (1) to characterize alkaline phosphatase (AP) activity in bovine oocytes and embryos with intact or removed zona pellucida (ZP); and (2) to evaluate the effect of culture medium constituents on AP activity. Alkaline phosphatase activity in non-matured and matured oocytes was most evident nearest the plasma membrane and perivitelline space. In more than 90% of two- to 16-cell embryos, AP activity was observed in the perivitelline space and at blastomere contacts. In blastocysts, AP activity was localized to the trophectoderm. Only after immunodissection was AP activity detected in the inner cell mass. Removal of the ZP by pronase or mechanical means reduced AP activity. Alkaline phosphatase activity was detected in evacuated ZP after mechanical removal. Specific constituents comprising the embryo culture medium altered AP activity. Alkaline phosphatase activity was reduced in eight- to 16-cell embryos and evacuated ZP cultured in CR1aa + 0.4% bovine serum albumin compared with embryos cultured in CR1aa alone or embryos co-cultured on a monolayer of Buffalo rat liver cells in the presence of 10% fetal bovine serum. The presence of AP activity at blastomere contacts and in evacuated ZP limits its usefulness as a marker for the differentiation of embryonic cells comprising the early cleavage-stage embryo.

Morphological characterization of skeletal cells in Cbfa1-deficient mice

To clarify the mechanisms by which core-binding factor-α1 (Cbfa1), an essential transcription factor in osteogenesis, functions in osteoblast matrix formation, as well as in chondrocyte differentiation and osteoclastic bone resorption, Cbfa1-deficient embryonic mice were investigated ultrastructurally and histocytochemically at 18.5 days postcoitum. In homozygotic mice, both endochondral and intramembranous ossification were arrested, although bone tissue had already formed at this stage in the wild type. The tibiae of homozygotic mice were characterized by calcified cartilage and alkaline phosphatase (ALP)-positive perichondrium, whereas membranous structures indicating the presence of ALP activity in the lateral portion were observed in the calvariae, rather than the bone tissue. Most of the ALP-positive perichondrial cells in homozygotic tibiae possessed a spindle-shaped cell contour and small cytoplasm, the extracellular matrix of which contained neither type I collagen nor calcifying matrix vesicles. In contrast, some perichondrial cells at the very middle part of tibiae became flattened. In the vicinity of these cells, a thin layer of type I collagen-based calcified matrix, containing osteopontin, bone sialoprotein, or osteocalcin, was observed. In the cartilage of mutant mice, we observed a hypoplasic zone of proliferative chondrocytes, the flattening of hypertrophic chondrocyte-like cells, and calcified chondrocytes which, while not degraded, did display a high level of cell function. Mononuclear osteoclastic cells were found in the perichondrium, near calcified chondrocytes, in mutant mice. Multinuclear osteoclasts possessing H +-ATPase and ruffled borders were also present, although only in limited numbers. Neither the development of ruffled borders nor intracellular polarization was complete. Because the majority of osteogenic cells in Cbfa1-deficient mice can neither form nor calcify the bone matrix, Cbfa1 principally plays essential roles in osteoblastic differentiation and bone matrix formation. Cbfa1 also affects both the proliferation and the differentiation of chondrocytes, whereas its absence prevents normal osteoclast formation and related functions.

Sodium butyrate-induced liver-type alkaline phosphatase activity in a small intestinal epithelial cell line, IEC6.

Sodium butyrate is a well-recognized differentiating agent inducing alkaline phosphatase activity, one of the epithelial differentiation markers. When IEC6 cells, a nontransformed, small intestinal epithelial cell line, were cultured with butyrate, this substrate induced alkaline phosphate activity in a time- and dose-dependent fashion. However, the type of isoenzyme involved was a liver-type, not an intestinal-type. Electron microscopy revealed that the induced activity was strictly localized in the cytosol and not on the plasma membrane. However, disaccharidase activities, another kind of differentiation marker, were also enhanced by sodium butyrate. In addition, the positive cells demonstrating the presence of alkaline phosphatase activity were preferentially observed in tubular structures. These data show that butyrate-induced alkaline phosphatase activity is closely associated with differentiation-like phenomena in IEC6 cells, although the type of isoenzyme and cellular localization of the activity are different from those observed in mucosa.

Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.

To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.

Production of fetuses from totipotent cultured bovine inner cell mass cells

Stem cells were derived from hatched blastocyst-stage mouse embryos of the C57BL/6 strain employing a knockout serum replacement instead of the traditional fetal calf serum, thereby avoiding the use of immunosurgery. Although fetal calf serum was not good for isolation of stem cells, a combination of this serum plus knockout serum increased the expansion rate of the cell culture. The derived cells were capable of maintaining an undifferentiated state during several passages, as demonstrated by the presence of alkaline phosphatase activity, stage-specific embryonic antigen 1 (SSEA-1), and octamer binding protein 4 (Oct-4). Suspension culture in bacteriological dishes gave better results than the hanging drop method for differentiation by means of embryoid body formation. Mouse embryonic stem cells showed spontaneous differentiation into derivatives of the 3 germ layers in culture media supplemented with fetal calf serum but not with knockout serum.

The proto-oncogene c-myc is involved in cell differentiation as well as cell proliferation: studies on growth plate chondrocytes in situ.

A combination of immunocytochemistry and microdensitometry has been used to localize and quantify the expression of the proto-oncogene c-myc within chondrocytes of the proximal growth plates of rat and chick long bones. Although the c-myc protein was localized in all chondrocytes of the growth plate of both species the most intense staining was restricted to the proliferating and differentiating chondrocytes. These were identified by their ability to synthesize DNA (bromodeoxyuridine positive) and the presence of alkaline phosphatase activity, respectively. Species differences did exist with the c-myc concentration of the chick proliferating and differentiating chondrocytes being higher (128% and 240%, respectively) than the respective chondrocytes of the rat. The higher c-myc concentration in the chick proliferating chondrocytes paralleled the differences in the bromodeoxyuridine labelling index between the two species. In the rat, the concentration of c-myc protein present in the differentiating chondrocytes was 74% higher than in the respective proliferating chondrocytes, while in the chick it was 146% higher. The data not only provides further evidence for a role of the c-myc protein in cell proliferation but also suggests involvement of this protein in chondrocyte differentiation and/or hypertrophy.

Studies of matrix vesicle-induced mineralization in a gelatin gel

Matrix vesicles isolated from fourth-pasage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and β-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicle is to transport enzymes for matrix modification.

Corticosteroid-induced changes in the responsiveness of human osteoblast-like cells to parathyroid hormone

We investigated the in vitro effect of corticosteroids on the responsiveness of human cells of osteoblast lineage to parathyroid hormone (PTH). Prior to corticosteroid treatment, the cells demonstrated only a small increase in cAMP production and no measurable change in transmembrane potential in response to PTH. Exposure of cells to dexamethasone resulted in a 5-fold increase in PTH-induced cAMP production and in measurable PTH-induced membrane depolarization in all cells studied. The effect of corticosteroids on cAMP production was specific for PTH (not seen with PGE 1 or forskolin), occurred in a time- and dose-dependent fashion and in the absence of cell proliferation. Most of the cells were of osteoblast lineage as determined by the presence of alkaline phosphatase activity and BGP secretion. These findings further support the idea that corticosteroids increase the sensitivity of cells of osteoblast lineage to PTH, perhaps by transforming cells which initially have a low responsiveness to PTH to a state of high responsiveness.

Ultracytochemical localization of alkaline phosphatase activity in endothelial cells in chronic relapsing experimental allergic encephalomyelitis.

To investigate the functions of endothelial cells (ECs) in chronic relapsing experimental allergic encephalomyelitis (EAE), we examined ECs ultracytochemically in various stages of EAE, in conjunction with the localization of alkaline phosphatase (AP) activity. We also studied the relation between the specific localization of AP activity and pathological features at each stage. Chronic relapsing EAE was induced in strain-13 guinea pigs by inoculation with homologous myelin. Controls were inoculated with complete Freund's adjuvant. The controls showed AP activity on the luminal and abluminal surfaces of the plasmalemma, and in pinocytic vesicles and vesicular pits. The localization of AP activity in the preclinical stage of EAE was similar to that in control animals. The initial inflammatory and actively demyelinating stage with perivascular cuffs of mononuclear cells showed AP-positive reactions on the abluminal surface of the plasmalemma, and in vesicles and pits, but not on the luminal surface in many ECs. In a later stage showing relatively old plaques with perivascular accumulation of debris-containing macrophages, AP activity continued to show localization similar to that seen in the initial stage, except for the presence of AP activity on some segments of the abluminal plasmalemma. Inactive lesions with marked perivascular fibrosis showed no AP reaction products. AP activity in unaffected areas showed the same localization as that in control animals throughout the various clinical stages of EAE. These findings suggest that AP activity decreased as the inflammatory demyelination in EAE progressed. The gradual disappearance of AP activity suggests development of functional impairment of ECs.