Showing papers on "Alkaline phosphatase published in 1993"

Phosphoprotein phosphatase activities in Alzheimer disease brain.

Abstract: Microtubule-associated protein tau is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of tau to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated tau could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of tau might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P-labeled phosphorylase kinase and poly(Glu, Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of tau in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of beta-amyloid by augmenting the amyloidogenic pathway processing of beta-amyloid precursor protein.

Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease

Abstract: We measured serum bone alkaline phosphatase (B-ALP) with a new immunoradiometric assay (IRMA) in a large sample of healthy controls comprising 173 women and 180 men, 20-88 yr of age, and in patients with metabolic bone disease. Using serum samples from patients with liver disease and patients with Paget's disease with elevated total alkaline phosphatase (T-ALP) as a source of, respectively, liver and bone isoenzymes, we determined a liver cross-reactivity of the IRMA of 16% that was confirmed by electrophoresis of the circulating alkaline phosphatase isoenzymes. The IRMA was linear for serial sample dilutions, the recovery ranged from 89-110%, and the intra- and interassay variations were below 7% and 9%, respectively. B-ALP increased linearly with age in both sexes, and the mean B-ALP serum levels were not significantly different for women and men (11.3 +/- 4.8 ng/mL for women; 11.0 +/- 4.0 ng/mL for men). The increase in B-ALP after the menopause was significantly higher than that in T-ALP (+77% vs. +24%; P < 0.001). When the values of postmenopausal women were expressed as the SD from the mean of premenopausal women, the mean Z scores were 2.2 +/- 1.8 for B-ALP and 0.9 +/- 1.3 for T-ALP (P < 0.001 between the two). Serum B-ALP was increased from control values in patients with Paget's disease (n = 57; mean, 171.8 +/- 135.6 ng/mL; P < 0.001), in patients with primary hyperparathyroidism (n = 18; mean, 17.2 +/- 5.9 ng/mL; P < 0.001), and in patients with chronic renal failure on hemodialysis (n = 83; mean, 36.6 +/- 35.7 ng/mL; P < 0.001). In patients with Paget's disease, B-ALP was highly correlated with T-ALP (r2 = 0.94; P < 0.001), and the decrease in its serum level was larger than that in T-ALP after treatment with the bisphosphonate pamidronate (-58% vs. -43%; P < 0.03). In patients with various liver diseases, B-ALP was slightly increased, but stayed within the normal range (mean +/- 2 SD) until T-ALP did not exceed 4.5 mu katal/L. We conclude that this new IRMA for B-ALP is reliable, has a low cross-reactivity with the liver isoenzyme, and appears to be more sensitive than T-ALP for the clinical investigation of patients with osteoporosis and other metabolic bone diseases.

Osteoblasts are target cells for transformation in c-fos transgenic mice.

Abstract: We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone-forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c-fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.

Butyrate Rapidly Induces Growth Inhibition and Differentiation in HT-29 Cells'

Abstract: Selected G1 events associated with butyrate-induced differentiation were examined in HT-29 colon adenocarcinoma cells. [3H]Thymidine incorporation by HT-29 cells was decreased to 40% of control levels by treatment with 5 mM butyrate for 24 h, and cell numbers decreased to 21% of control levels after 48 h of treatment. Cells released from butyrate arrest entered S phase approximately 24 h after release, and serum-deprived HT-29 cells escaped growth inhibition if butyrate was added 8 h or more after serum restimulation. Northern analysis of RNA isolated from rapidly growing HT-29 cells showed a marked induction of alkaline phosphatase mRNA expression within 12 h of treatment with 5 mM butyrate. The appearance of alkaline phosphatase mRNA was temporally associated with a 5-fold increase in expression of transforming growth factor beta 1 (TGF-beta 1) mRNA. Expression of the nuclear protooncogene c-myc began to decrease 30 min after treatment with butyrate and was decreased 4.5-fold 4 h after treatment; however, expression of other immediate-early genes (nup/475 and zif/268) was not significantly affected. Histochemical staining of HT-29 monolayers showed that no cells were positive for alkaline phosphatase protein prior to treatment, and 90% were positive 48 h after treatment. TGF-beta 1 and TGF-beta 2 had no effect on HT-29 cell growth. TGF-beta 1 did not induce alkaline phosphatase mRNA or histochemical positivity. These data indicate that butyrate arrests HT-29 cell growth early in G1.(ABSTRACT TRUNCATED AT 250 WORDS)

Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.

Abstract: Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

Effect of omeprazole, an inhibitor of H+,K(+)-ATPase, on bone resorption in humans

Abstract: Omeprazole is an inhibitor of gastric H+, K+-ATPase. Although the major proton transport of osteoclast is mediated by a vacuolar-type H+-ATPase which is different from the gastric H+, K+-ATPase,in vitro studies have demonstrated that omeprazole inhibits bone resorption. In this study, the effect of omeprazole on bone resorption was evaluated in patients who had a history of gastric ulcer and were treated with maintenance doses of H2 blocker without any gastric complaints at the study time. H2 blocker administration was changed to omeprazole treatment in the study group and to no treatment in the control group. Urinary excretion of hydroxyproline and calcium decreased after omeprazole treatment in the study group. Serum intact PTH, alkaline phosphatase, osteocalcin, and tartrate-resistant acid phosphatase (TRAP) increased in this group. In the control group, there were not any changes in these parameters. The discrepancy between serum TRAP and urinary excretion of hydroxyproline and calcium in the study group was thought to be due to the suppression of bone resorption by omeprazole, which probably interfered the acidification at resorption lacunae and resulted in the inactivation of TRAP and other lysosomal enzymes. The results of our study suggest the possibility that the specific inhibitors of the osteoclastic proton pump (such as bafilomycins) will more effectively suppress bone resorption and be useful for the treatment of metabolic bone diseases with increased bone resorption.

Growth hormone stimulates proliferation and differentiation of normal human osteoblast-like cells in vitro

Abstract: In this study we investigated the direct, short-term effects of human growth hormone (hGH) on the biology of normal adult human osteoblast-like (hOB) cells cultured from trabecular bone explants. In Subconfluent cultures, hGH stimulated hOB proliferation in a dose-dependent fashion (P < 0.001, n = 15) with half-maximal effects at a concentration of 10 ng/ml. These mitogenic effects were detectable within 24 hours as shown by bromodeoxyuridine labeling. In confluent cultures containing mainly quiescent cells, hGH increased levels of alkaline phosphatase (P < 0.05, n = 10) and to a lesser degree levels of procollagen type I carboxyterminal propeptide (PICP) (P = 0.07, n = 9). Effects on osteocalcin (bone GLa protein, BGP) levels were highly variable among different cell strains and only 7 of 10 cell strains showed a stimulatory response (P = 0.16). We also studied the effects of hGH on osteoblastic production of insulin-like growth factor I (IGF-I) and IGF-II as well as the production of GH-dependent, insulin-like growth factor binding protein 3 (IGFBP-3). Under basal conditions, human osteoblasts produced IGF-II and IGFBP-3 in the conditioned medium. When stimulated with hGH, minor insignificant increase in both IGF-II and IGFBP-3 (125% and 126% of control, respectively) were detectable. No IGF-I was detectable in the conditioned medium under basal conditions or after stimulation with hGH. In conclusion, the results obtained in this study suggest that GH exerts direct anabolic effects on human osteoblasts.

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells.

Abstract: A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley-Liss, Inc.

Plasma bone-specific alkaline phosphatase as an indicator of osteoblastic activity

Abstract: The total plasma alkaline phosphatase level has long been recognised as an indicator of osteoblastic activity, but lack of specificity makes it an insensitive index of the progress of disease and the response to treatment. Selective precipitation by wheatgerm lectin allows measurement of the plasma bone-specific alkaline phosphatase. We measured the plasma levels of this isoenzyme in 170 normal Chinese adolescents and adults, in 49 adults with fractures of a long bone, in 15 patients with osteosarcoma and in 38 patients with osteolytic metastases. The enzyme activity was also determined in 39 patients with liver disease. Of the patients with fractures, 94% had increased plasma activity during the healing process. The level was also increased in those with osteosarcoma but not in those with osteolytic bone metastases. There was no significant increase in activity in the patients with liver disease. We conclude that the plasma bone-specific alkaline phosphatase activity is a sensitive and reliable measure of osteoblastic activity.

Two cell lines from bone marrow that differ in terms of collagen synthesis, osteogenic characteristics, and matrix mineralization.

Abstract: Two cloned cell lines were isolated from cultures of mouse bone-marrow cells. One of the lines, D1, exhibited osteogenic properties and synthesized type-I collagen (alpha 1)2 alpha 2. The second cell line, D2, was not osteogenic and produced a collagen homotrimer (alpha 1)3. Whereas the extracellular matrix of the D1 cell cultures contained striated collagen fibrils, presumably composed of type-I collagen, the homotrimer-producing D2 cells did not demonstrate striated collagen fibrils. Instead, they had thin filaments without detectable striations. Sodium ascorbate stimulated collagen synthesis at the transcriptional level in both the D1 and the D2 cells. The bone-producing characteristics of D1 in vitro included high levels of alkaline phosphatase, increased cyclic adenosine monophosphate on treatment with parathyroid hormone, and expression of osteocalcin mRNA. The D1 cells, unlike the D2 cells, produced a mineralized matrix in vitro. Mineralization in the cultures of the D1 cells occurred in nodules of increased cell density, which also contained the cells with the highest concentrations of collagen mRNA, as shown by in situ hybridization. When the D1 cells were implanted in a diffusion chamber in vivo, a mixture of both osteogenic and adipogenic tissues was formed. This indicates that the D1 cell line is derived from an early marrow stromal precursor that is multipotential.

Multiple Forms of Human Serum Alkaline Phosphatase: Detection and Quantitation

Abstract: The measurement of serum alkaline phosphatase (EC 3.1.3.1 ALP) has been popular in clinical practice for over 50 years despite a limited knowledge of the structure, function and pathophysiology of this enzyme family. The existence of multiple forms of the enzyme has provided a great deal of opportunity for research and this has helped to refine ideas on the clinical utility of measurement of the enzymes in serum as well as to elucidate their structure and function. 1,2

Multicenter Evaluation of Iso-ALP Test Kit for Measurement of Bone Alkaline Phosphatase Activity in Serum and Plasma

Abstract: A test kit (Iso-ALP, Boehringer Mannheim) for measuring human bone alkaline phosphatase activity in serum or plasma was evaluated in five laboratories in three countries. The assay is based on the principle described by Rosalki and Foo (Clin Chem 1984;30:1182-6) and uses wheat germ lectin to precipitate bone alkaline phosphatase. Residual ALP in the supernate in comparison with total ALP is used to quantify the bone fraction. The imprecision of residual ALP measurement was low (median between-run CV 4.9%) and comparable with that of total ALP. Linearity of precipitation was demonstrable up to a bone ALP activity (diethanolamine buffer 37 degrees C) of 2000 U/L, though a matrix effect was observed for dilutions of high-activity sera in saline or bovine serum albumin. For assaying patients' samples, different batches of lectin demonstrated excellent comparability. Taking electrophoresis as a basis for standardization, we determined that the lectin precipitated approximately 90% of bone ALP, but < 5% of nonbone ALP. From this we derived serum/plasma upper reference limits for bone ALP activity in adults and children.

Human bone marrow stromal cells express an osteoblastic phenotype in culture.

Abstract: This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.

Bone sialoprotein (BSP) secretion and osteoblast differentiation : relationship to bromodeoxyuridine incorporation, alkaline phosphatase, and matrix deposition

Abstract: We defined two distinct maturational compartments (proliferative and secretory) of osteogenic cells in vivo on the basis of ALP activity, BrdU incorporation, cell shape, and BSP production. BSP immunoreactivity was found to mark cells in the secretory but not in the proliferative compartment. We established the phenotypic similarity of primitive marrow stromal cells with proliferating perichondral cells (fibroblast-like, ALP+, BrdU+, BSP-). This suggests the potential functional equivalence of the two cell types as committed non-secretory osteogenic cells and points to the duality of osteogenic cell compartments as a generalized feature of bone formation. We further showed that although BSP secretion is a hallmark of the onset of osteogenesis, BSP antigenicity is lost both in osteoid and in a large proportion of mature osteoblasts during subsequent phases of bone deposition. This suggests that bone formation may not be a uniform event, as bone cells actually deposit antigenically, and likely biochemically, distinct matrices at specific times.

Vitamin K-induced changes in markers for osteoblast activity and urinary calcium loss

Abstract: The objective of this study was to identify subjects in whom vitamin K has an effect on markers for calcium and bone metabolism and to detect hitherto-unnoticed correlations between vitamin K-induced changes in these markers. Participants in our studies were apparently healthy women, in whom we measured serum-immunoreactive osteocalcin (irOC) before and after adsorption to hydroxylapatite; total serum alkaline phosphatase (T-AP) and bone-specific alkaline phosphatase (B-AP); and fasting urinary calcium and creatinine. We describe a trial among 145 women who were treated with vitamin K (1 mg/day) for 2 weeks, and a prospective placebo-controlled trial among two groups each of 70 postmenopausal women with a treatment period of 3 months. It turned out that in elderly women vitamin K induced increased levels of serum irOC with a high affinity for hydroxylapatite (irOCbound), whereas that with low affinity (irOCfree) remained unaffected. In placebo-treated women the ratio irOCfree/irOCbound shifted from 0.38 to 0.65 around the 50th year of age. This shift was not found in vitamin K-treated women. After 3 months of treatment the vitamin K-induced changes in irOCbound were correlated with changes in B-AP, whereas irOCfree was correlated to urinary calcium excretion. In fast losers of urinary calcium vitamin K induced a 30% decrease of calcium excretion. The hypothesis is put forward that irOCbound may be a marker for bone formation, that serum irOCfree may be a marker for bone resorption, and that the serum irOCfree/irOCbound ratio may become a marker for skeletal remodeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline phosphatase staining of pig and sheep epiblast cells in culture

Abstract: To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis.

Analysis of the topology of a membrane protein by using a minimum number of alkaline phosphatase fusions.

Abstract: An approach to analyzing the topology of membrane proteins with alkaline phosphatase fusions is described. Precise fusions were constructed by using polymerase chain reaction at the C terminus of each hydrophilic region of the membrane protein. The disruption of topogenic signals is thereby minimized, and predictable anomalous results are avoided. The Escherichia coli MalG protein has been analyzed.

Immunohistochemical markers of carcinoma in situ of the testis also expressed in normal infantile germ cells.

Abstract: Carcinoma in situ of the testis is an intratubular, pre-invasive lesion preceding germ cell tumour. In adult men, carcinoma in situ cells differ in several aspects from normal germ cells. For example, placental-like alkaline phosphatase and/or the epitopes for the monoclonal antibodies M2A, 43-9F and TRA-1-60 are not seen in normal germ cells, whereas their presence is considered a specific sign of carcinoma in situ. As it is known that placental-like alkaline phosphatase and the epitope for TRA-1-60 are expressed in normal fetal germ cells it is possible that the markers could appear in normal infantile germ cells in a period after birth before they lose their expression. In children, carcinoma in situ cells may be difficult to identify morphologically and the use of the markers could be of great value. However, little information is available on the expression of the markers of adult carcinoma in situ in normal infantile germ cells. We investigated gonads from 66 boys less than 15 years old who died suddenly. Their deaths were unrelated to testicular disease. Immunohistochemical staining with anti-placental-like alkaline phosphatase antibody and monoclonal antibodies TRA-1-60 and 43-9F were performed. We found that these markers were expressed in some normal infantile germ cells until the age of 1 year. Therefore, these markers are not suitable for diagnosis of carcinoma in situ during the early postnatal period of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex-dependent effects of 17-beta-estradiol on chondrocyte differentiation in culture

Abstract: This study examined the effects of 17-beta-estradiol (E2) on chondrocyte differentiation in vitro. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used to determine whether the effects of E2 were dependent on the stage of chondrocyte maturation and whether they were sex-specific. [3H]-incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators of differentiation. Alakaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. Specificity of the E2 effects was assessed using 17-alpha-estradiol. The role of fetal bovine serum and phenol red in the culture medium was also addressed. The results demonstrated that E2 decreases cell number and [3H]-incorporation in female chondrocytes, indicating that it promotes differentiation of these cells. Alkaline phosphatase specific activity is stimulated in both growth zone and resting zone cells, but the effect is greater in the less mature resting zone chondrocytes. The increase in enzyme activity is targeted to the matrix vesicles in both cell types, but the fold increase is greater in the growth zone cells. In male chondrocytes, there was a decrease in [3H]-incorporation at high E2 concentrations in resting zone cells at the earliest time point examined (12 hours) and a slight stimulation in alkaline phosphatase activity in growth zone cells at 24 hours. Cells cultured in serum-free medium exhibited a dose-dependent inhibition in alkaline phosphatase activity when cultured with E2, even in the presence of phenol red. E2-stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. E2 increased percent collagen production in female cells only; the magnitude of the effect was greatest in the resting zone chondrocyte cultures. The results of this study indicate that the effects of E2 are dependent on time of exposure, presence of serum, and the sex and state of maturation of the chondrocytes. E2-stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles. © 1993 Wiley-Liss, Inc.

Culture and differentiation of chondrocytes entrapped in alginate gels.

Abstract: We studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.

Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis

Abstract: Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)