acid-phosphatase and thiazolyl-blue

To investigate 1α,25-(OH)₂D₃ regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1α,25-(OH)₂D₃ during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1α,25-(OH)₂D₃ inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1α,25-(OH)₂D₃ administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.

Topics: Acid Phosphatase; Animals; Blotting, Western; Calcitriol; Calcium Channel Agonists; Cell Differentiation; Cell Line; Cell Proliferation; Gene Expression Regulation, Enzymologic; Isoenzymes; Matrix Metalloproteinase 9; Mice; Osteoclasts; Tartrate-Resistant Acid Phosphatase; Tetrazolium Salts; Thiazoles

The prophylactic effects of Hijikia fusiforme on bone metabolism were examined using in vitro indices of bone formation and resorption. As the indices of bone formation, osteoblast proliferation and differentiation were measured through mitochondrial enzyme activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] and bone marker alkaline phosphatase (ALP) activity. The aqueous extract of H. fusiforme stimulated the proliferation of the human osteoblast-like cell line MG63 and the ALP activity of the mouse osteoblast-like cell line MC3T3-E1. Moreover, extracellular matrix mineralization was accelerated by the addition of H. fusiforme. As the indices of bone resorption, differentiation of the murine macrophage/osteoclast precursor cell line RAW 264.7 by receptor activator of nuclear factor-κB ligand (RANKL) was measured as tartrate-resistant acid phosphatase-positive multinucleated cells, which were suppressed by H. fusiforme. Additionally, H. fusiforme lowered the secreted amount of RANKL that is required for the osteoclastic differentiation and activation, but the amount of osteoprotegerin as a decoy receptor for RANKL was not affected. The bone-protective effects of H. fusiforme in estrogen-deficient ovariectomized rats were also investigated. Osteoporosis was induced in female Sprague-Dawley rats by ovariectomy for 15 weeks, and then H. fusiforme was orally administered at a dose of 100  mg/kg of body weight every day for 6 weeks. Bone mineral density in the group orally administered H. fusiforme was increased, compared with ovariectomized rats, but not significantly (P>.05). Oral administration of H. fusiforme improved microarchitecture of bone in terms of bone volume (bone volume/total volume ratio) and trabecular separation (P<.05). The amounts of osteocalcin and C-telopeptide type I collagen in serum were measured as the biomarkers for bone formation and resorption. The level of osteocalcin in serum was increased, but not significantly. However, the level of C-telopeptide type I collagen in serum was significantly decreased (P<.05). When the results are taken together, the present study indicates that H. fusiforme might be useful in the treatment of osteoporosis.

Topics: Acid Phosphatase; Administration, Oral; Alkaline Phosphatase; Animals; Biomarkers; Bone Density; Bone Resorption; Cell Differentiation; Cell Line; Cell Proliferation; Collagen Type I; Female; Humans; Isoenzymes; Mice; Osteocalcin; Osteoclasts; Osteogenesis; Osteoporosis; Ovariectomy; Peptides; Phaeophyceae; RANK Ligand; Rats; Rats, Sprague-Dawley; Tartrate-Resistant Acid Phosphatase; Tetrazolium Salts; Thiazoles

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 μM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.

Topics: Acid Phosphatase; Aeromonas salmonicida; Analysis of Variance; Animals; Gadus morhua; In Vitro Techniques; Kidney; Leukocytes; Oligodeoxyribonucleotides; Peroxidase; Respiratory Burst; Tetrazolium Salts; Thiazoles; Vibrio

Environmental pollutants such as heavy metals exert immunotoxic effects on aquatic organisms. The immune defence of molluscs is comprised of cell-mediated and humoral mechanisms, in which haemocytes play a key role. In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of zinc in vitro. Cells were exposed for 24 h to ZnCl(2) concentrations of 0, 10, 100 or 1000 microM. The effects of zinc on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Immunotoxicity was reflected by a significant decrease in the number of viable haemocytes (LC(50)(24 h) = 314 microM). Moreover, the cell area was dramatically reduced, and the percentage of rounded cells increased with increasing zinc concentrations. Exposure to 1000 muM zinc induced a significant reduction in acid phosphatase activity, phagocytic activity and reactive oxygen species production in haemocytes. However, several haemocyte parameters increased significantly after 24 h of zinc exposure. In response to a 1000 microM exposure, the phenoloxidase level was 26-fold higher than that of the control, and non-specific esterase activity was increased by 69% above that of the control. These results suggest a relationship between zinc exposure and alterations in the functional responses of haemocytes from H. tuberculata.

Topics: Acid Phosphatase; Animals; Cells, Cultured; Dose-Response Relationship, Drug; Environmental Pollutants; Esterases; Flow Cytometry; France; Gastropoda; Hemocytes; In Vitro Techniques; Lethal Dose 50; Models, Immunological; Monophenol Monooxygenase; Phagocytosis; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Zinc

The genomic DNA of Escherichia coli, which contains the unmethylated CpG motif, was used to evaluate the immunostimulating effect of bacterial DNA on innate immune responses in the bivalve mussel Hyriopsis cumingii Lea. The results showed that the E. coli DNA had no significant effect on the production of superoxide anion (O(2-)) or acid phosphatase (AP) by haemocytes in vitro. However, the bactericidal activity of the haemocytes was significantly increased when the cells were incubated with 50 or 100mug/ml bacterial DNA for 12 and 24h. Antibacterial activity, lysozyme activity, and prophenoloxidase (proPO) production of haemolymph were also increased, when the bivalve molluscs were injected with 50 or 100mug/ml of bacterial DNA for 12 and 24h. These activities returned to the control level after 48h. This work showed the bacterial DNA with unmethylated CpG motif could activate some parameters of the immune system of bivalve molluscs in vivo and in vitro.

Topics: Acid Phosphatase; Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Catechol Oxidase; DNA, Bacterial; Enzyme Induction; Enzyme Precursors; Escherichia coli; Hemocytes; Hemolymph; Immunity, Innate; Lipopolysaccharides; Muramidase; Superoxides; Tetrazolium Salts; Thiazoles; Time Factors; Unionidae

In the central nervous system (CNS), tumor necrosis factor-alpha (TNF-alpha) derived from activated microglia plays a critical role as an inflammatory mediator. In this study, we examined the function of TNF-alpha as an autocrine mediator in microglial activation. TNF-alpha induced TNF-alpha production by microglia through ligation of TNF receptor 1 (TNFR1). TNF-alpha also increased the production of other inflammatory mediators. The activation of microglia by lipopolysaccharide is partially mediated by microglia-derived TNF-alpha. These findings suggest the existence of a positive feedback loop in the activation of microglia via TNF-alpha. This autocrine loop may be involved in the prolonged activation of microglia.

Topics: Acid Phosphatase; Animals; Animals, Newborn; Antibodies; Autocrine Communication; Blotting, Northern; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Microglia; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha

The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L: -tyrosine derivatives 1-3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treatment with the derivatives 1-3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1-3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acid Phosphatase; Apoptosis; Bone and Bones; Bone Diseases, Metabolic; Caspase 3; Caspases; Cell Nucleus; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Isoenzymes; Models, Chemical; NF-kappa B; Osteoblasts; Osteoclasts; Piperazines; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2X7; Tartrate-Resistant Acid Phosphatase; Tetrazolium Salts; Thiazoles; Time Factors

The present study examined the effect of ambroxol on free radical production, granule enzyme release, and cell death in silica-activated rat alveolar macrophages. The action of ambroxol was assayed by measuring changes in the activities of protein kinase C (PKC) and tyrosine kinase (PTK) and in the intracellular calcium level. Ambroxol attenuated the production of superoxide, hydrogen peroxide, and nitric oxide and the release of acid phosphatase and lysozyme in macrophages activated by silica. Staurosporine, genistein, EGTA, and trifluoperazine inhibited the silica-induced free radical production and granule enzyme release. Silica induced the increase in PKC and PTK activities and the elevation of intracellular calcium level in macrophages, which was decreased by ambroxol. Silica induced a cell death and increased the caspase-3 activity in macrophages in a concentration-dependent manner. Ambroxol decreased the silica-induced cell viability loss in macrophages. The results show that ambroxol decreases the stimulated responses and cell death in rat alveolar macrophages exposed to silica, which may be accomplished by inhibition of activation processes, protein kinases, and calcium transport. The inhibitory effect of ambroxol on silica-induced cell death appears to provide the protective effect on pulmonary tissues against the toxic action of silica.

Topics: Acid Phosphatase; Ambroxol; Animals; Calcium; Caspase 3; Caspases; Cell Death; Cytoplasmic Granules; Expectorants; Hydrogen Peroxide; In Vitro Techniques; Macrophage Activation; Macrophages, Alveolar; Muramidase; Nitrites; Oxidants; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Respiratory Burst; Silicon Dioxide; Superoxides; Tetrazolium Salts; Thiazoles

The effect of thiram, a fungicide that increases the incidence of tibial dyschondroplasia (TD) in poultry, was studied in vitro using growth plate chondrocyte culture. Thiram caused a significant reduction in alkaline phosphatase, acid phosphatase, and lactate dehydrogenase (LDH) activities at concentrations of 5 microM and above. It was highly cytotoxic to chondrocytes at and above this concentration as determined by their ability to reduce 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (triazolyl blue, MTT), a marker of cellular viability. An increase in the leakage of LDH into culture media was evident at concentration as low as 1 microM. Very few differences were noticed in the electrophoretic migration profiles of cell-extract proteins at any treatment level relative to control. The cytotoxic effect of thiram is possibly due to its damaging effect on the cell membrane, which may be responsible for chondrocyte death.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Blotting, Western; Cell Death; Cell Survival; Cells, Cultured; Chickens; Coloring Agents; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Growth Plate; L-Lactate Dehydrogenase; Molecular Weight; Oxidation-Reduction; Tetrazolium Salts; Thiazoles; Thiram

A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.

Topics: Acid Phosphatase; Animals; Cell Survival; Cells, Cultured; Colorimetry; Culture Media, Serum-Free; Fibroblast Growth Factor 2; Mice; Mice, Inbred Strains; Neurons; Tetrazolium Salts; Thiazoles