Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The technique of two-dimensional gel electrophoresis was used for analysis of tyrosine phosphorylated polypeptide substrates after epidermal growth factor (EGF)-induced stimulation of receptor tyrosine kinase activity in a brush border fraction of human placental syncytiotrofoblast cells. After incubation with [gamma 32P]ATP, followed by autoradiography of the gels, 35 phosphorylated components were detected, of which 8 were strongly tyrosine phosphorylated by EGF. Using a more sensitive assay with phosphotyrosine-specific antibody, an additional 12 polypeptide components were found to be strongly tyrosine phosphorylated by EGF. A number of the phosphorylated substrates could be aligned with components in a protein catalog of the human brush border membrane fraction that was characterized by glycoprotein staining, Triton X-114 fractionation, immunoreaction with specific antibodies, and comigration with 35S-labeled AMA (transformed human amnion) cells. Identified components, stimulated by EGF, in addition to well-recognized substrates (calpactin II, ezrin, EGF receptor) included beta-tubulin and serum albumin, while other cytoskeletal proteins and alkaline phosphatase were excluded as substrates. A notable feature of the catalog was that a number of glycoproteins were present in both the membrane and cytoskeletal fraction, suggesting involvement in membrane/cytoskeletal interactions. The data demonstrate the feasibility of using two-dimensional gel electrophoresis in a global way to identify target substrates for tyrosine kinase activity. In addition they suggest that many of these are located in the vicinity of tyrosine kinase at the membrane/cytoskeletal border at a location which is probably involved, at the molecular level, in morphological changes of the plasma membrane associated with cell proliferation.
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PMID:Application of two-dimensional gel analysis to identification and characterization of tyrosine phosphorylated substrates for growth factor receptors. 768 72

To determine whether the poorly differentiated AMOC-2 human ovarian adenocarcinoma cell line was capable of undergoing differentiation, AMOC-2 cells were exposed to 2 mM sodium butyrate, 2.5% dimethylsulfoxide, or 4 mM dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP) for 6 days. These treatments resulted in growth inhibition, a reduction in clonogenicity and an increase in cellular glycogen content. Significant increases in heat stable alkaline phosphatase activity also occurred after exposure to sodium butyrate. In addition, a thorn-like microfilament structure observed in untreated cells was diminished concomitantly with morphological changes that included flattening, enlargement and extended cytoplasmic processes after exposure to sodium butyrate or dibutyryl cAMP. Furthermore, treatment with sodium butyrate increased the intracellular concentrations of beta-tubulin, vimentin, neurofilaments (M(r) 210,000) and cytokeratin (M(r) 56,000-58,000). These changes were completely reversed after removal of the inducing agent. The findings suggest that treatment of AMOC-2 cells with sodium butyrate induced a more differentiated phenotype, although terminal differentiation was not achieved.
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PMID:Effects of sodium butyrate, dimethylsulfoxide and dibutyryl cAMP on the poorly differentiated ovarian adenocarcinoma cell line AMOC-2. 830 43

After direct photoaffinity cross-linking of [3H]GTP to the beta-subunit of tubulin, followed by tryptic digestion and alkaline phosphatase treatment, we employed cis-diol-specific boronate gel chromatography and reversed-phase high-pressure liquid chromatography to purify a peptide containing most of the covalently bound radioactivity. The sequence of this peptide corresponded to that of residues 3-19 of beta-tubulin. Residue 10 of the peptide, which is Cys-12 in beta-tubulin, could not be identified. The fast atom bombardment mass spectrum of this peptide showed the presence of a predominant species with a molecular mass of 2022 kDa (2021 kDa for the 12C variant), which is 255 Da greater than the molecular mass of the peptide. Fast atom bombardment collision-activated decomposition mass spectrometry analysis produced fragments which are consistent with the beta(3-19) peptide but having a unit of mass of 358 at position 12. Thermolysin digestion of the tryptic peptide restricted the cross-linking site to the 9-amino acid sequence, I(L)QAGQXGNQ. The molecular mass of this peptide was 1174 kDa, which is equal to the mass of the beta(7-15) peptide containing an extra group of mass 255. To explain the molecular masses of the two labeled peptides, which are 26 atomic mass units less than expected, a mechanism of photolabeling is proposed that involves opening of the guanine ring and loss of the C-6 carbonyl function as CO2.
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PMID:Exchangeable GTP binding site of beta-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling. 841 20

The glial cytoplasmic inclusion (GCI) is a histological hallmark for multiple system atrophy (MSA). These inclusions are in oligodendrocytes, contain microtubular structures of 20-30 nm diameter, and can be labelled immunohistochemically with antibodies to ubiquitin, alphaB-crystallin, alpha- and beta-tubulin, and the microtubule-associated protein tau. GCIs have been compared with neuronal inclusions in other neurodegenerative disorders including the neurofibrillary tangles (NFTs) found in Alzheimer's disease (AD), which also contain tau protein. In order to determine whether the tau protein of GCIs in MSA is similar to that observed in AD we used a panel of antibodies to phosphorylation-independent (SMI51, TP007, TP70), dephosphorylation-dependent (Tau.1), and phosphorylation-dependent antibodies to tau and neurofilaments (AT8, AT180, AT270, SMI31, SMI34, RT97, BF10, 8D8). Immunohistochemistry was performed on paraffin wax-embedded brain tissue of the cerebellum, brainstem, and frontal lobes (Brodmann areas 4/6) of ten clinically and neuropathologically well-characterised cases of MSA, two cases of AD, and two normal controls. The NFTs of the AD cases were labelled with all the phosphorylation-dependent and phosphorylation-independent antibodies and with Tau.1 only after treatment with alkaline phosphatase. In contrast, GCIs were immunolabelled by the phosphorylation-independent antibodies and Tau.1, but not by the phosphorylation-dependent antibodies. These data demonstrate that the tau in GCIs is different from the abnormally phosphorylated tau found in AD and is similar to normal adult tau. The mechanism causing the abnormal accumulation of tau in GCIs remains to be elucidated.
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PMID:Tau protein in the glial cytoplasmic inclusions of multiple system atrophy can be distinguished from abnormal tau in Alzheimer's disease. 925 61

Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.
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PMID:Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy. 1051 Nov 6

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro. Furthermore, these derived cells tended to neurally differentiate in vitro, especially under high-density culture conditions. We successfully isolated neural progenitor cells from differentiating hEG cultures and about 10% cells induced by 2microM all-trans-retinoic acid (RA) or 0.1mM dibutyryl cyclic AMP (dbcAMP)/1mM forskolin to mature neurons expressing microtubule-associated protein 2ab (MAP2ab), synaptophysin, beta-tubulin III, neuron-specific enolase (NSE), tyrosine hydroxylase (TH), but no glial fibrillary acid protein (GFAP) and choline acetyl transferase (ChAT). The data suggested that hEG cells may provide a potential source of cells for use in transplantation therapy for neurological degenerative diseases.
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PMID:In vitro neuronal differentiation of cultured human embryonic germ cells. 1562 48

Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two-step culture in 10 and 70% serum-supplemented media for 5 days. Such multipotent MG, called promicroglioblasts (ProMGBs), formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared to be the source of NG2+ ProMGB aggregates. The aggregates were MG marker+/NG2+/GFAP+/NCAM+/S-100beta- and had alkaline phosphatase activity. The marked accumulation of NG2+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2+ MG in the wounds separated with trypsin-EDTA formed NG2+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing beta-tubulin III+ cells in vitro easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain.
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PMID:NG2 proteoglycan-expressing microglia as multipotent neural progenitors in normal and pathologic brains. 1653 76

We have investigated a novel ultrafine grained (UFG) Zr obtained by severe plastic deformation (SPD) which resulted in a refinement of the grain size by several orders of magnitude. Compared to conventional Zr, higher hardness values were measured on UFG Zr. Polished surfaces having similar topographical features from both materials were prepared, as assessed by atomic force microscopy (AFM). Surface hydrophobicity of Zr, evaluated by measuring water contact angles, was unaffected by grain size reduction. In vitro biocompatibility was addressed on conventional and UFG Zr surfaces and, for comparative purposes, a polished Ti6Al4V alloy was also investigated. Cell attachment and spreading, actin and beta-tubulin cytoskeleton reorganisation, fibronectin secretion and cellular distribution as well as cell viability were evaluated by culturing human osteoblastic Saos-2 cells on the surfaces. The osteoblastic response to conventional Zr was found to be essentially identical to Ti6Al4V and was not affected by grain size reduction. In order to evaluate the ability of the surfaces to promote osteogenic maturation and bone matrix mineralisation, human mesenchymal cells from bone marrow were switched to the osteoblastic phenotype by incubation in osteogenic induction media. Compared to undifferentiated mesenchymal cells, alkaline phosphatase activity and formation of mineralisation nodules were enhanced to the same extent on both Zr surfaces and Ti6Al4V alloy after induction of osteoblastic differentiation. In summary, improved mechanical properties together with excellent in vitro biocompatibility make UFG Zr a promising biomaterial for surgical implants.
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PMID:In vitro biocompatibility of an ultrafine grained zirconium. 1762 24

Molecular data for nephridial development in polychaetes are not available yet. The scope of our work was to establish a reference system for future investigations using two markers for nephridial development: beta-tubulin as marker for cilia and alkaline phosphatase (AP) activity for secretory epithelia. The markers identified, unexpectedly, three consecutively forming generations of nephridia: (1) a transitory unciliated, but AP-positive head kidney, (2) a transitory larval nephridium, which undergoes a morphological transition from a protonephridium to a funnelled nephridium concomitant with the development of the coelomic cavity and finally, (3) the serially arranged metanephridia. The spatial arrangement of larval and definitive nephridia, revealed an up to now unknown developmental boundary between the synchronously forming larval and the serially proliferating definitive segments. Development of three consecutive sets of nephridia with different morphology and biochemical properties was unexpected and reveals an interesting multistep process in the development of excretory structures in Platynereis.
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PMID:Three consecutive generations of nephridia occur during development of Platynereis dumerilii (Annelida, Polychaeta). 2054 33