Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dominant paradigm of protein engineering is structure-based site-directed mutagenesis. This rational approach is generally more effective for the engineering of local properties, such as substrate specificity, than global ones such as allostery. Previous workers have modified normally unregulated reporter enzymes, including beta-galactosidase, alkaline phosphatase, and beta-lactamase, so that the engineered versions are activated (up to 4-fold) by monoclonal antibodies. A reporter that could easily be "reprogrammed" for the facile detection of novel effectors (binding or modifying activities) would be useful in high throughput screens for directed evolution or drug discovery. Here we describe a straightforward and general solution to this potentially difficult design problem. The transcription factor p53 is normally regulated by a variety of post-translational modifications. The insertion of peptides into intrinsically unstructured domains of p53 generated variants that were activated up to 100-fold by novel effectors (proteases or antibodies). An engineered p53 was incorporated into an existing high throughput screen for the detection of human immunodeficiency virus protease, an arbitrarily chosen novel effector. These results suggest that the molecular recognition properties of intrinsically unstructured proteins are relatively easy to engineer and that the absence of crystal structures should not deter the rational engineering of this class of proteins.
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PMID:Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors. 1611 6

A Down's syndrome associated gene, Single Minded 2 gene short form (SIM2-s), is specifically expressed in colon tumors but not in the normal colon. Antisense inhibition of SIM2-s in a RKO-derived colon carcinoma cell line causes growth inhibition, apoptosis, and inhibition of tumor growth in a nude mouse tumoriginicity model. The mechanism of cell death in tumor cells is unclear. In the present study, we investigated the pathways underlying apoptosis. Apoptosis was seen in a tumor cell-specific manner in RKO cells but not in normal renal epithelial cells, despite inhibition of SIM2-s expression in both of these cells by the antisense. Apoptosis was depended on WT p53 status and was caspase-dependent; it was inhibited by a pharmacological inhibitor of mitogen-activated protein kinase activity. Expression of a key stress response gene, growth arrest and DNA damage gene (GADD)45alpha, was up-regulated in antisense-treated tumor cells but not in normal cells. In an isogenic RKO cell line expressing stable antisense RNA to GADD45alpha, a significant protection of the antisense-induced apoptosis was seen. Whereas antisense-treated RKO cells did not undergo cell cycle arrest, several markers of differentiation were deregulated, including alkaline phosphatase activity, a marker of terminal differentiation. Protection of apoptosis and block of differentiation showed a correlation in the RKO model. Our results support the tumor cell-selective nature of SIM2-s gene function, provide a direct link between SIM2-s and differentiation, and may provide a model to identify SIM2-s targets.
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PMID:Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells. 1612 20

Our study focused on investigating the mechanism of action of estrogen in regulating p53 levels within osteoblasts. In the studies reported here, we attempted to understand the role of estrogen receptors, ER-alpha and ER-beta, in the regulation of p53 and osteoblast differentiation. We stably expressed ER-alpha and ER-beta in ROS 17/2.8 cells and isolated several single cell clones. These clones were initially characterized for expression of the exogenous receptors, and representative clones from each type were chosen for further analyses. Cell proliferation, alkaline phosphatase activity, and the viability of these clones in culture were tested. The cells expressing exogenous ER-alpha exhibited more differentiated characteristics than cells expressing ER-beta. Morphologically, ER-beta-overexpressing cells were more rounded than the ER-alpha-overexpressing cells, which were more elongated and fibroblastic in appearance. The ER-beta-expressing cells had a higher survival and growth rate when compared with ER-alpha cells. The ER-alpha clones were not as viable as ER-beta clones, and some of the ER-alpha cell lines showed signs of senescence, with an increase in senescence-associated (SA) galactosidase activity. The basal levels of p53 functional activity were higher in cells expressing ER-alpha as was protein expression of the p53-regulated gene p21. The significance of these receptors to osteoblast differentiation and p53 regulation is discussed.
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PMID:Effect of overexpression of estrogen receptors in osteoblasts. 1640 12

Aims-To develop a protocol that is applicable to single strand conformation polymorphism (SSCP), direct sequencing and loss of heterozygosity analysis of DNA.Methods-The protocol is based on the detection of biotinylated DNA by a Streptavidin-alkaline phosphatase conjugate. Biotinylation of DNA was achieved by using 5'-end biotinylated primers for PCR. After polyacrylamide gel electrophoresis, the DNA fragments were transferred to a nylon membrane by contact blotting. Depending on the alkaline phosphatase substrate, DNA was visualised either colorimetrically or by chemiluminescence.Results-The method was verified by the identification and characterisation of p53 mutations by SSCP analysis and direct DNA sequencing, as well as the assessment of DNA loss in human lung carcinomas by microsatellite polymorphism allelotyping.Conclusions-The protocol is simple, does not require specialised equipment and would be particularly useful for laboratories with experience in Streptavidin-biotin methodology.
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PMID:Use of non-radioactive detection in SSCP, direct DNA sequencing and LOH analysis. 1669 52

Low-energy laser irradiation (LELI) accelerates wound healing and is thought to accelerate bone formation. However, the mechanism of laser healing is not clear. To clarify the biological mechanism of LELI healing, we investigated the effects of LELI on rat osteoblasts in vitro. Osteoblastic cells from 3-day-old Wistar rat calvaria were irradiated using a low-energy gallium-aluminum-arsenide (Ga-Al-As) diode laser. Bone formation, osteoblast differentiation, and cell proliferation were evaluated by von Kossa staining, reverse-transcription polymerase chain reaction, alkaline phosphatase (ALP) staining, 5-bromo-2'-deoxyuridine (BrdU) uptake, and fluorescence-activated cell sorter (FACS) analysis. At 21 days after LELI, the greatest bone formation was observed with irradiation energy of 3.75 J/cm2 and the first week after seeding. LELI (3.75 J/cm2) induced an increased number of cells at day 3. LELI-stimulated differentiation in osteoblastic cells was demonstrated by the increases of Runx2 expression and ALP-positive colonies. By contrast, at 1 day after laser irradiation, the number of cells in the irradiation group was significantly lower than that in the control group. BrdU uptake indicated lower proliferation 12 and 24 hours after irradiation compared with the control. Furthermore, FACS data demonstrated a higher proportion of cells in the G2/M phase of the cell cycle 12 hours after irradiation compared with the control. G2/M arrest was confirmed by the appearance of G2/M arrest marker 14-3-3-sigma or phospho-p53. These results demonstrate that LELI induces not only acceleration of bone formation but also initial G2/M arrest, which may cause wound healing like tissue repair.
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PMID:Optimal low-energy laser irradiation causes temporal G2/M arrest on rat calvarial osteoblasts. 1716 May 75

In vivo and in vitro studies indicate that a subpopulation of human marrow-derived stromal cells (MSCs, also known as mesenchymal stem cells) has potential to differentiate into multiple cell types, including osteoblasts. In this study, we tested the hypothesis that there are intrinsic effects of age in human MSCs (17-90 years). We tested the effect of age on senescence-associated beta-galactosidase, proliferation, apoptosis, p53 pathway genes, and osteoblast differentiation in confluent monolayers by alkaline phosphatase activity and osteoblast gene expression analysis. There were fourfold more human bone MSCs (hMSCs) positive for senescence-associated beta-galactosidase in samples from older than younger subjects (P < 0.001; n = 17). Doubling time of hMSCs was 1.7-fold longer in cells from the older than the younger subjects, and was positively correlated with age (P = 0.002; n = 19). Novel age-related changes were identified. With age, more cells were apoptotic (P = 0.016; n = 10). Further, there were age-related increases in expression of p53 and its pathway genes, p21 and BAX. Consistent with other experiments, there was a significant age-related decrease in generation of osteoblasts both in the STRO-1+ cells (P = 0.047; n = 8) and in adherent MSCs (P < 0.001; n = 10). In sum, there is an age-dependent decrease in proliferation and osteoblast differentiation, and an increase in senescence-associated beta-galactosidase-positive cells and apoptosis in hMSCs. Up-regulation of the p53 pathway with age may have a critical role in mediating the reduction in both proliferation and osteoblastogenesis of hMSCs. These findings support the view that there are intrinsic alterations in human MSCs with aging that may contribute to the process of skeletal aging in humans.
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PMID:Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts. 1824 63

We have established a novel production process which allows up to fourfold higher production of a model secreted protein, the human secreted alkaline phosphatase (SEAP), in Chinese hamster ovary (CHO) cells. A cytostatic production phase is established in which cell proliferation is inhibited or completely abolished. Such a cytostatic production phase is established by overexpression of the tumor suppressor genes p21, p27, or p53175P (a p53 mutant showing specific loss of apoptotic function) under transcriptional control of a tetracycline-repressible promoter (P(hCMV*-1)). In order to minimize complications due to possible clonal variation of selected, stable cell lines, our investigations are based on transiently transfected subpopulations, that have become a useful tool in industrial R&D. These subpopulations have been selected by flow cytometry for the expression of genes encoded on a dicistronic expression vector. These vectors contain a dicistronic expression unit consisting of the genes encoding the green fluorescent protein (GFP) or SEAP, followed by one of the cytostatic genes p21, p27 or p53175P encoded by the second cistron. p21, p27 as well as p53175P block the cell cycle of CHO cells in the G1-phase for a prolonged period. However, these G1-arrested cells remain viable and proliferation proficient upon repression of expression of the cytostatic gene. All three of the cytostatic genes studied provided similar regulation of proliferation, and also similar enhancements in SEAP production, suggesting that higher productivity may be a general and intrinsic feature of G1-phase arrested CHO cells. Overall productivity is most likely enhanced because growth-arrested cells do not need to devote cellular resources to biomass production.
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PMID:A novel cytostatic process enhances the productivity of Chinese hamster ovary cells. 1863 2

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells.
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PMID:Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line. 1883 54

p73 and p63 are members of the p53 gene family that play an important role in development and homeostasis, mainly by regulating transcription of a variety of genes. We report here that apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid transport proteins, is a direct transcriptional target of the p53 family member genes. We found that the expression of apoD was specifically up-regulated by either TAp73 or TAp63 but not significantly by p53. In addition, apoD transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abolishes induction of apoD transcription following cisplatin treatment. We also identified a p73/p63-binding site in the promoter of the apoD gene that is responsive to the p53 family members. The ectopic expression of TAp73 as well as the addition of recombinant human apoD to culture medium induced the osteoblastic differentiation of the human osteosarcoma cell line Saos-2, as assessed by alkaline phosphatase activity. Importantly, apoD knockdown abrogated p73-mediated alkaline phosphatase induction. Moreover, TAp73-mediated apoD expression was able to induce morphological differentiation, as well as expression of neuronal markers, in the human neuroblastoma cell line SH-SY5Y. These results suggest that apoD induction may mediate the activity of p73 in normal development.
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PMID:p53 family members regulate the expression of the apolipoprotein D gene. 1900 18

T-box (Tbx)3, a known transcriptional repressor, is a member of a family of transcription factors, which contain a highly homologous DNA binding domain known as the Tbx domain. Based on the knowledge that mutation of the Tbx3 gene results in limb malformation, Tbx3 regulates osteoblast proliferation and its expression increases during osteoblast differentiation, we predicted that Tbx3 is an important regulator of osteoblast cell functions. In this study, we evaluated the consequence of transgenic overexpression of Tbx3 on osteoblast differentiation. Retroviral overexpression increased Tbx3 expression >100-fold at the mRNA and protein level. Overexpression of Tbx3 blocked mineralized nodule formation (28 +/- 8 vs. 7 +/- 1%) in MC3T3-E1 cells. In support of these data, alkaline phosphatase (ALP) activity was reduced 33-70% (P < 0.05) in both MC3T3-E1 cells and primary calvaria osteoblasts overexpressing Tbx3. In contrast, Tbx3 overexpression did not alter ALP activity in bone marrow stromal cells. Tbx3 overexpression blocked the increase in expression of key osteoblast marker genes, ALP, bone sialoprotein, and osteocalcin that occurs during normal osteoblast differentiation, but had little or no effect on expression of proliferation genes p53 and Myc. In addition, Tbx3 overexpression abolished increased osterix and runx2 expression observed during normal osteoblast differentiation, but the change in Msx1 and Msx2 expression over time was similar between control and Tbx3 overexpressing cells. Interestingly, osterix and runx2, but not Msx1 and Msx2, contain Tbx binding site in the regulatory region. Based on these data and our previous findings, we conclude that Tbx3 promotes proliferation and suppresses differentiation of osteoblasts and may be involved in regulating expression of key transcription factors involved in osteoblast differentiation.
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PMID:T-box 3 negatively regulates osteoblast differentiation by inhibiting expression of osterix and runx2. 1911 50


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