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Showing papers on "Alkaline phosphatase published in 1996"


Journal ArticleDOI
TL;DR: Two isoforms (B56β and B56δ) are highly expressed in adult brain; here it is shown that mRNA for these isoforms increases severalfold when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment.

384 citations


Journal ArticleDOI
TL;DR: Results indicate that the inherent osteogenic ability of marrow stromal stem cells in pore regions of HA can be stimulated using tissue culture technology; and thus, formed osteogenic HA can show immediate osteoblastic activity in in vivo situations, suggesting the applicability of the HA in clinical situations.
Abstract: We employed culture technology, which provides bone tissue in vitro, to expand and promote the osteogenic ability of marrow cells in porous hydroxyapatite (HA). Marrow cells were obtained from rat femur and cultured in a standard medium for 10 days, then trypsinized to make composites of HA and the cells. An additional 2-week culture (subculture) was done for the composite in a standard medium with or without the addition of dexamethasone (Dex). The 2-week subcultured composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity and bone Gla protein content, as well as histological analysis of decalcified and undecalcified sections. In Dex-treated composites, high alkaline phosphatase activity could be detected 1 week after implantation and was maintained until 8 weeks after implantation. The bone Gla protein content could also be detected 1 week after implantation, followed by a steady increase with the passage of time until 8 weeks after implantation. The histological analysis showed active bone formation even 1 week after implantation. The bone formation was evidenced by active osteoblast lining and the appearance of calcein labeling following calcein injection 1 week after implantation. Thus, Dex-treated subcultured marrow cells in pore regions of HA showed a high osteogenic response immediately after transplantation. In contrast, Dex-untreated composite did not show bone formation and contained traces of these biochemical parameters. These results indicate that the inherent osteogenic ability of marrow stromal stem cells in pore regions of HA can be stimulated using tissue culture technology; and thus, formed osteogenic HA can show immediate osteoblastic activity in in vivo situations, suggesting the applicability of the HA in clinical situations.

271 citations


Journal ArticleDOI
TL;DR: It is shown that OP-1 acts on early stage mesenchymal progenitor cells (ATDC5, C3H10T1/2) to induce chondroblastic differentiation, while OP- 1 strongly enhances the osteoblastic phenotype of committed osteoblasts (MC3T3-E1), possibly explaining its induction of the endochondral ossification cascade in vivo.

255 citations


Journal ArticleDOI
TL;DR: Plasma bAP level equal or higher than 20 ng/mL, either alone or combined with plasma iPTH of 200 pg/ mL, had the highest sensitivity, specificity, and predictability values for the diagnosis of high-turnover bone disease, and formally excluded patients with normal or LTBD.
Abstract: Plasma total versus bone alkaline phosphatase as markers of bone turnover in hemodialysis patients. Plasma bone-specific alkaline phosphatase (bAP) has been demonstrated to be more reliable than total alkaline phosphatases (tAP) in providing information about bone turnover in patients with metabolic bone diseases. This study surveyed 42 hemodialysis patients who underwent a systematic transiliac bone biopsy for histomorphometry study. Plasma bAP was determined by using a new immunoassay (Tandem-R Ostase, Hybritech, Liege, Belgium). Plasma bAP values were compared with those of two other plasma markers of bone metabolism, namely tAP and intact parathyroid hormone (iPTH), for the correlations with bone histomorphometric parameters. Patients with high-turnover bone disease (HTBD) (N = 32) had significantly higher plasma bAP levels than patients with normal or low bone turnover (N/LTBD) (N = 10) (66.9 +/- 63.5 ng/mL versus 10.8 +/- 4.2 ng/mL, respectively). Bone formation and resorption were highly correlated in these patients, and plasma bAP levels were positively correlated with bone resorption parameters, including osteoclast surface (r = 0.39, P

246 citations


Journal ArticleDOI
TL;DR: It is suggested that bone alkaline phosphatase, the closer physiological link with osteoblast function and the lesser expense for its determination, is a useful tool in the noninvasive diagnosis of the adynamic type of bone disease in the individual patient.
Abstract: Background. Adynamic bone disease was recently described to be increasingly prevalent in the dialysis population. At present the diagnosis of this type of renal osteodystrophy can only be made by bone histomorphometry. We assessed the value of different biochemical serum markers in the diagnosis of adynamic bone disease. Methods. In 103 haemodialysis patients a bone biopsy was performed after double tetracycline labelling, and the serum levels of intact PTH, osteocalcin, and the bone isoenzyme of alkaline phosphatase were determined. Bone alkaline phosphatase was measured by an optimized agarose gel electrophoretic method, recently shown to have a high accuracy, precision and reproducibility, also in the lower range. Results. In 38 (37%) of the patients the diagnosis of adynamic bone disease was histologically established. Constructing receiver operator curves optimal cut-off levels for the diagnosis of adynamic bone disease were determined, being ≤27 U/litre for the bone isoenzyme of alkaline phosphatase, ≤14 μg/litre for osteocalcin and ≤150 pg/ml for intact PTH. Concentrations of bone alkaline phosphatase or intact PTH below these cut-off levels, were shown to be the best performing tests in the detection of adynamic bone disease as indicated by a sensitivity of 78.1 and 80.6% and a specificity of 86.4 and 76.2% respectively. Applying Bayes' theorema, it was calculated that in the current haemodialysis population in which a prevalence of adynamic bone disease up to 35% has been described, the positive predictive values for the proposed cut-off values are 75% for bone alkaline phosphatase, 65% for intact PTH and 55% for osteocalcin. Moreover, in this population, levels of bone alkaline phosphatase and intact PTH below the optimal cut-off excluded hyperparathyroid bone disease. Conclusion. In view of the relative easy and accurate methodology for bone alkaline phosphatase determination, the closer physiological link with osteoblast function and the lesser expense for its determination we suggest that this marker is a useful tool in the noninvasive diagnosis of the adynamic type of bone disease in the individual patient.

185 citations


Journal ArticleDOI
TL;DR: The activation energy of the reaction catalyzed by a single molecule is determined with high precision and the most active molecules have over a 10-fold higher activity than the least active molecules.
Abstract: Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; micr...

171 citations


Journal ArticleDOI
TL;DR: Results suggest that the L- selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-α.

149 citations


Journal ArticleDOI
TL;DR: Results indicate that ions associated with Ti-6Al-4V alloy inhibited the normal differentiation of bone marrow stromal cells to mature osteoblasts in vitro, suggesting that ions released from implants in vivo may contribute to implant failure by impairing normal bone deposition.

146 citations


Journal ArticleDOI
TL;DR: Treatment-related alterations of clinical chemistry and histopathology occurred frequently in this series of toxicity studies in rats, indicating that clinical chemistry evaluations can be useful for detecting potential treatment effects throughout a study.

134 citations


Journal Article
TL;DR: When tested in chronic colitis (2 and 4 weeks), quercitrin treatment decreased colonic damage score and the incidence of diarrhea, and normalized the colonic fluid transport, and all other parameters were unaffected.
Abstract: Quercitrin was tested for acute and chronic anti-inflammatory activity in trinitrobenzenesulfonic acid-induced rat colitis. The inflammatory status was evaluated by myeloperoxidase, alkaline phosphatase and total glutathione levels, leukotriene B4 synthesis, in vivo colonic fluid absorption, macroscopical damage and occurrence of diarrhea and adhesions. Treatment with 1 or 5 mg/kg of quercitrin by the oral route reduced myeloperoxidase and alkaline phosphatase levels, preserved normal fluid absorption, counteracted glutathione depletion and ameliorated colonic damage at 2 days. Increasing or lowering the dose of the flavonoid resulted in marked loss of effect. The acute anti-inflammatory effect of quercitrin is unrelated to impairment of neutrophil function or lipoxygenase inhibition, and it may be caused by mucosal protection or enhancement of mucosal repair secondary to increased defense against oxidative insult and/or preservation of normal colonic absorptive function. When tested in chronic colitis (2 and 4 weeks), quercitrin treatment (1 or 5 mg/kg.day) decreased colonic damage score and the incidence of diarrhea, and normalized the colonic fluid transport. All other parameters were unaffected. The chronic effect of the flavonoid is apparently related to its action on colonic absorption, although it can be partly secondary to its acute beneficial effect.

124 citations


Journal ArticleDOI
01 Jan 1996-Gene
TL;DR: This article summarizes the current knowledge on the transcription of the genes encoding acid and alkaline phosphatases and the inorganic phosphate transporter of Saccharomyces cerevisiae and describes its application for the study of regulatory mechanisms.

Journal ArticleDOI
TL;DR: The results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast.
Abstract: PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activ...

Journal ArticleDOI
TL;DR: The combination of immortalized rat osteoblasts and the ALP activity test provides a powerful tool for in vitro testing of orthopaedic materials.

Journal ArticleDOI
TL;DR: It is concluded that penetration of the blood‐brain barrier by morphine‐6‐glucuronide may depend on the expression of the product of the multidrug‐resistance (MDR) gene in brain capillary endothelial cells.
Abstract: 1. Morphine-6-glucuronide is one of the major metabolites of morphine. The potent analgesic action of this compound together with its potential lower apparent toxicity in man, when compared with morphine, indicated its clinical importance. 2. Primary cultures of porcine brain capillary endothelial cells were used to study brain penetration of morphine-6-glucuronide. Biochemical characterization of the cell cultures revealed a marked enrichment in enzymatic activity of alkaline phosphatase (56 fold) and angiotensin converting enzyme (230 fold) as compared to whole brain tissue. By immunostaining the presence of vimentin, factor VIII, the tight junction associated protein ZO-1, and P-glycoprotein was shown. Functional characterization revealed that the carrier system responsible for transport of neutral amino acids was intact. 3. Uptake and transport of morphine-6-glucuronide was marginal and in the range of the extracellular marker sucrose. However, uptake of morphine-6-glucuronide was enhanced significantly (P < 0.0001) in presence of the inhibitors of P-glycoprotein, verapamil or vincristine. The finding that morphine-6-glucuronide may serve as a substrate for P-glycoprotein was confirmed in multidrug-resistant P388 tumour cells. 4. We conclude that penetration of the blood-brain barrier by morphine-6-glucuronide may depend on the expression of the product of the multidrug-resistance (MDR) gene in brain capillary endothelial cells.

Journal ArticleDOI
TL;DR: The modification of the cytomegalovirus promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described.
Abstract: The FLAG peptide, AspTyrLysAspAspAspAspLys, has been used as an epitope tag in a variety of cell types. The modification of the cytomegalovirus (CMV) promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described. As a functional test, the bacterial alkaline phosphatase gene was cloned into both vectors, and anti-FLAG monoclonal antibodies were used for detection of FLAG epitope-tagged bacterial alkaline phosphatase in mammalian cells. In addition, secreted bacterial alkaline phosphatase was purified from the extracellular medium by anti-FLAG affinity chromatography.

Journal ArticleDOI
TL;DR: The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during bone cell differentiation and exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy.
Abstract: Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation To that end, transgenic mice were produced using a 29-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization The 2T3 cells als

Journal ArticleDOI
TL;DR: Results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP, and in most other clinical situations, serum TAP appears to provide sufficient clinical information.
Abstract: To evaluate the diagnostic validity of new assays for bone-specific alkaline phosphatase (BAP), we compared measurements of total alkaline phosphatase (TAP) in serum with results for three different assays of serum BAP in healthy adults (n = 119), patients with chronic nonskeletal disorders (n = 123), and patients with metabolic bone diseases (n = 113) Serum TAP was determined by a standard colorimetric assay, BAP by the methods of lectin precipitation (L-BAP), enzyme immunoassay (E-BAP), and immunoradiometric assay (I-BAP) Impairment of liver function resulted in significant increases of all alkaline phosphatase (AP) measurements, with the smallest changes being exhibited by E-BAP Compared with the results by TAP, diagnostic sensitivity (ie, of values exceeding the reference interval) was not improved by BAP, but receiver-operating characteristic (ROC) curve analyses revealed improved discrimination for primary hyperparathyroidism by E-BAP These results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP In most other clinical situations, serum TAP appears to provide sufficient clinical information; however, the cross-sectional study design used here allows no statement about the usefulness of BAP in serial measurements

Journal ArticleDOI
TL;DR: A direct inhibitory effect of ethanol on osteoblast proliferation without overt cellular toxicity is confirmed that may, in part, explain the reduced bone mass observed in those who consume excessive amounts of alcohol.
Abstract: The habitual consumption of alcoholic beverages is clearly associated with low bone mass and an increased prevalence of skeletal fractures. Microscopic analysis of skeletal tissue from alcoholic patients reveals reduced osteoblast number and suppressed bone formation activity with a relative sparing of resorptive indices. The decreased number of osteoblasts observed in alcoholic subjects results from either impaired proliferation or accelerated senescence. Polyamines and ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, are essential for cell proliferation in a variety of cell types. To determine if the adverse effect of ethanol on osteoblast number involves modulation of polyamine biosynthesis, we examined the effect of ethanol on parameters of cell growth and ODC activity in a human osteoblast-like osteosarcoma cell line (TE-85). Ethanol markedly impaired DNA synthesis and cell proliferation in a dose-dependent fashion, but alkaline phosphatase activity (a marker of differentiated osteoblast function) remained intact, and accelerated apoptosis was not evident. Thus, the reduced osteoblastic cell number was a result of a direct effect on proliferative processes rather than a nonspecific toxic effect of ethanol to accelerate cell death. Induction of ODC activity was impaired in ethanol-exposed cell cultures in a dose-dependent fashion that paralleled the antiproliferative effects. Finally, supplemental polyamine administration substantially improved DNA synthesis in ethanol-exposed UMR 106-01 cell cultures. These data confirm a direct inhibitory effect of ethanol on osteoblast proliferation without overt cellular toxicity that may, in part, explain the reduced bone mass observed in those who consume excessive amounts of alcohol.

Journal ArticleDOI
TL;DR: The observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex‐induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells.
Abstract: Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10(-10) M with maximum values achieved at 10(-8)M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1 ,25(OH)(2)D(3) during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells.

Journal ArticleDOI
TL;DR: The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells.
Abstract: We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinhardtii in response to phosphorus deprivation. Phosphorus-deprived cells increase extra-cellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the Alkaline phosphatase activity. This phosphatase has optimal activity at pH 9.5 and a Km of 120 to 262 microns for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells.

Journal ArticleDOI
TL;DR: It is suggested that a suppressed activity of osteoblasts may contribute to osteoporosis and fractures in patients with vitamin B12 deficiency.
Abstract: Pernicious anemia has recently been recognized as one of the risk factors for osteoporosis and bone fractures, but the underlying pathophysiologic mechanism is still unknown. To determine whether vitamin B12 has any direct effect on osteoblasts, we studied the effects of vitamin B12 on the proliferation and alkaline phosphatase activity in human bone marrow stromal osteoprogenitor cells (hBMSC) and UMR106 osteoblastic cells. Vitamin B12 at concentrations as low as 10(-12) mol/L significantly stimulated [3H]-thymidine incorporation in both types of cells, but concentrations higher than 10(-12) mol/L did not produce a greater effect. Vitamin B12 in the concentration range from 10(-12) to 10(-8) mol/L concentration-dependently increased alkaline phosphatase activity in both hBMSC and UMR106 cells. Based on these results, we suggest that a suppressed activity of osteoblasts may contribute to osteoporosis and fractures in patients with vitamin B12 deficiency.

Journal Article
TL;DR: Results indicate that ellagic acid administration orally can circumvent the carbon tetrachloride toxicity and subsequent fibrosis in animals and rectified liver pathology.
Abstract: Chronic administration of carbon tetrachloride in liquid paraffin (1.7) ip; 0.15 ml, (20 doses) has been found to produce severe hepatotoxicity, as seen from the elevated levels of serum and liver glutamate-pyruvate transaminase, alkaline phosphatase and lipid peroxides. The chronic administration of carbon tetrachloride was also found to produce liver fibrosis as seen from pathological analysis as well as elevated liver-hydroxy proline. Oral administration of ellagic acid was found to significantly reduce the elevated levels of enzymes, lipid peroxide and liver hydroxy proline in these animals and rectified liver pathology. These results indicate that ellagic acid administration orally can circumvent the carbon tetrachloride toxicity and subsequent fibrosis.

Journal ArticleDOI
TL;DR: The studies suggest that differential expression of IGF-II and IGFBPs may be playing a critical role in both proliferation and differentiation of colonocytes, and suggested that CaCo2 cell differentiation may require an attenuation ofIGF-II effects.
Abstract: The extent to which the insulin-like growth factor (IGF) system contributes to the initiation and progression of colon cancer remains poorly defined. We recently reported that a majority of human colon cancers express and secrete the potent mitogen IGF-II and at least two inhibitory binding proteins, IGFBP-2 and IGFBP-4. In the present study we measured the expression and secretion of IGF-II, IGFBP-2, and IGFBP-4 in relation to growth and differentiation of CaCo2 human colon cancer cells, which undergo spontaneous enterocytic differentiation in culture. Under the conditions of the present study, CaCo2 cells demonstrated an initial rapid phase of growth between Day 2 through days 7-9 of culture, followed by a significant retardation in the growth between days 9-13. Alkaline phosphatase (ALP) activity, a marker of enterocytic differentiation, progressively increased between Days 7-13 in culture, temporally correlating with post-confluent phase of negligible growth. These changes in growth and differentiation were accompanied by > 80% decline in the relative concentration of IGF-II messenger RNA (mRNA) between Days 2-13. In contrast, the relative mRNA concentrations of inhibitory binding proteins (IGFBP-2 and IGFBP-4) increased rapidly to 200% of Day 2 values by Days 5-7 before returning to baseline levels by Day 13. The relative protein concentrations of the three factors measured in the conditioned media of the cells followed a pattern very similar to that measured for the mRNA levels. While the changes in the relative protein concentrations and mRNA levels of IGF-II and IGFBP-4 were statistically significant, the changes measured in the RNA and protein levels of IGFBP-2 were not, as a result of large inter experimental variations. Thus these results suggested that CaCo2 cell differentiation may require an attenuation of IGF-II effects. To confirm the latter possibility, additional studies were conducted with a specific neutralizing antibody against IGF-II. Incubation of CaCo2 cells with anti-IGF-II antibodies from Day 0 through Day 7 significantly retarded the growth of the cells and was accompanied by a significant increase in the concentration of Alkaline phosphatase activity per 10(6) cells. Recently, we reported a potent inhibitory role of IGFBP-4 in the growth of colon cancer cells. In the present studies, a possible important role of IGF-II is illustrated not only in the growth but also in the differentiation of colonic cells. Our studies thus suggest that differential expression of IGF-II and IGFBPs may be playing a critical role in both proliferation and differentiation of colonocytes.

Journal ArticleDOI
TL;DR: The findings indicate that S.b. boulardii has a positive effect on the maturation of enterocytes and only a minor influence on lymphocytes and duodenal mucosa.
Abstract: Saccharomyces boulardii (Sb) is used for the prevention and treatment of diarrhea of different etiologies We prospectively investigated the effects of Sb on lymphocytes and duodenal mucosa Before and after oral administration of Sb for 3 weeks, circulating and intestinal lymphocytes were isolated and characterized by flow cytometry Trophic effects on duodenal mucosa were investigated by morphometry and determination of brush border enzyme activity Results were compared intraindividually before and after Sb In intestinal lymphocytes no phenotypic changes were observed CD4+ cells of the peripheral blood had a significantly increased expression of CD25 (p < 002) None of twelve volunteers had an increase in villous surface area (ns) Immunoglobulin A content in small intestine secretion was unaltered An increase in brush border enzyme activity of lactase, α-glucosidase, and alkaline phosphatase was observed (p < 001) Our findings indicate that Sb has a positive effect on the maturation of enterocytes and only a minor influence on lymphocytes

Journal ArticleDOI
TL;DR: In these patients with active RA, bone resorption was increased, while bone formation was within normal limits, and the effect of corticosteroid pulse treatment on bone may be assumed to be relatively mild.
Abstract: OBJECTIVE: To examine the effect of high dose corticosteroid pulse treatment (three times 200 mg dexamethasone intravenously in eight days) on calcium and bone metabolism in 17 consecutive patients with active rheumatoid arthritis (RA). METHODS: Bone formation was quantified by measurement of serum alkaline phosphatase, osteocalcin, and carboxyterminal propeptide of type I procollagen (pro-I-CPP) concentrations. Bone resorption was measured by urinary excretion of calcium, hydroxyproline, (free and total) deoxypyridinoline (Dpyr), (free and total) pyridinoline (Pyr), and serum concentrations of the carboxyterminal cross linked telopeptide of type I collagen (I-CTP). Disease activity of RA was measured by erythrocyte sedimentation rate, C reactive protein, and Ritchie and Thompson joint scores. RESULTS: Disease activity was initially high, and decreased during corticosteroid pulse treatment and the following five weeks. Osteocalcin, alkaline phosphatase, and pro-I-CPP concentrations were initially within normal limits, while I-CTP, Dpyr, and Pyr were increased. Osteocalcin and pro-I-CPP concentrations decreased (p < 0.01) during corticosteroid pulse treatment, but rapidly returned to baseline after the treatment. No changes were observed in alkaline phosphatase and urinary excretion of calcium and hydroxyproline. Bone resorption measured by serum I-CTP and urinary excretion of Pyr and Dpyr was unchanged or decreased (p < 0.05-0.01), depending on the time of measurement and the parameter measured. CONCLUSIONS: In these patients with active RA, bone resorption was increased, while bone formation was within normal limits. During high dose corticosteroid pulse treatment, bone formation was only transiently decreased, while markers of bone resorption were unchanged or decreased. Because corticosteroid pulse treatment has only a short term negative effect on bone formation, and because it probably reduces bone resorption, at least partly as a result of the decreased disease activity, the effect of corticosteroid pulse treatment on bone may be assumed to be relatively mild.

Journal ArticleDOI
TL;DR: It is concluded that phosphatase activities in gingival crevicular fluid may be a useful means for monitoring tissue responses to orthodontic treatment.

Journal ArticleDOI
TL;DR: The results suggest that tension-force may affect PDL metabolism, depending on the functional role of ALP, and the finding of inhibited ALP activity in response to tension- force was consistent with the observation that L/B/K ALP mRNA levels were decreased in responseto cyclic tension-forces.
Abstract: Alkaline phosphatase (ALP) activity is involved in the process of calcification in various mineralizing tissues, and it is found at much higher levels in the periodontal ligament (PDL) than in other connective tissues. Since the PDL lies between hard tissues and functions as a cushion mitigating mechanical stress, such as occlusal and orthodontic forces, this stress may modulate ALP activity in PDL cells, which themselves may affect adjacent alveolar bone metabolism. The objective of this study was to determine the level of ALP activity and the gene expression of liver/bone/kidney (L/B/K) ALP in human PDL fibroblasts in response to cyclic tension-forces. Human PDL cells were cultured on flexible-bottomed plates and placed on a Flexercell Strain Unit. Cells were flexed at 6 cycles/min (5 sec strain, 5 sec relaxation) at 6 levels of tension-force (9%, 12%, 15%, 18%, 21%, and 24% increase in surface area) for 5 days. There was no significant difference in cell proliferation between the cells subjected to the...

Journal ArticleDOI
TL;DR: Fusions between Lpp′OmpA and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane, showing consistency with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors.
Abstract: The Lpp′OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusions between Lpp′OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp′OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp′OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (β-lactamase, alkaline phosphatase, Cex cellulase, Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp′OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

Journal ArticleDOI
TL;DR: The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenousTenascin helps to maintain the functional state of cultured osteoblast-like cells.
Abstract: The extracellular matrix protein tenascin is secreted by osteoblasts but absent from mineralized bone matrix. The current study was undertaken to test the hypothesis that tenascin regulates osteoblast behaviour. Three osteoblast-like cell lines UMR-106, ROS-17/2.8 (rat) and SAOS-2 (human) were used to investigate the role of tenascin in osteoblast morphology, differentiation and proliferation. Two of three cell lines adhered specifically to tenascin, remaining round and failing to spread. Tenascin as a substratum stimulated alkaline phosphatase activity (a marker of osteoblast differentiation) in two of three cell lines. Moreover, anti-tenascin in the medium caused a reduction in alkaline phosphatase levels in all three cell lines. Anti-tenascin also inhibited collagen synthesis, an important osteoblast function. Since it seemed possible that tenascin may exert its effects on cell function through its ability to cause cell rounding, the ability of cell shape change alone to influence alkaline phosphatase levels was investigated. Cells were incubated in the presence of cytochalasin D and alkaline phosphatase levels assayed. Alkaline phosphatase activity was not elevated by cytochalasin D treatment, indicating that cell rounding alone is insufficient to mimic the effect of tenascin. Anti-tenascin caused a slight increase in proliferation of SAOS-2 cells, indicating that tenascin is itself inhibitory. In ROS 17/2.8 and UMR-106 cells, in contrast, proliferation was inhibited by anti-tenascin. The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenous tenascin helps to maintain the functional state of cultured osteoblast-like cells.

Journal ArticleDOI
TL;DR: Data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2, and other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals.
Abstract: We investigated the involvement of CYP1A2 in the pharmacokinetics and metabolism of caffeine using mice lacking its expression (CYP1A2 -/-). The half-life of caffeine elimination from blood was seven times longer in the CYP1A2 -/- than wild-type mice. The clearance was concomitantly eight times slower. No parameter that could affect the pharmacokinetics differed between CYP1A2-/-and wild-type mice such as creatinine for kidney function; alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin for liver function; or albumin for protein binding. Other P450s CYP2A, 2B, 2C, 2EI, and 3A were also unchanged in the knockout animals. Caffeine 3-demethylated metabolites thought previously to be characteristic of CYP1A2 (especially 1-methylxanthine and I-methylurate) were also found in the urines of the CYP1A2-/-animals, although at 40% of the level found in wild-type mice. These data indicate that the clearance of caffeine in wild-type mice is primarily determined by CYP1A2.