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)

Immunohistochemical analysis of intermediate filament (IF) proteins was performed on frozen sections of 16 childhood glial tumors using a library of 10 antigen-specific IF protein directed monoclonal antibodies (MoABs) and a four-step biotin-streptavidin-alkaline phosphatase conjugated antigen detection immunocytochemical technique. Human glial fibrillary acidic protein (GFAP) and vimentin were expressed in all brain tumors. High molecular weight (200 kDa) neurofilament (NF-H) protein was expressed in 15 out of 16 tumors; medium molecular weight (160 kDa) neurofilament (NF-M) in seven out of 16 tumors; and low molecular weight (68 kDa) neurofilament (NF-L) in five out of 16. Positive acidic keratin reactivity was found in five out of 16 tumors using MoAB AE1. Expression of a keratin pair was detected with MoAB AE2 in five out of 16 tumors. A second keratin pair in 14 out of 16 glial tumors was demonstrated with MoAB AE3. Immunostaining with AE5 defined the expression of another basic keratin (64 kDa) in nine out of 16 glial tumors. Finally, in 14 out of 16 astrocytomas an individual 51 kDa acidic keratin (detected with MoAB AE8) was expressed. Glial tumor cells contain cell lineage specific and nonspecific IF proteins in the following IF pattern: AE3+, AE8+, GFAP+, vimentin+, and NF-H+. The heterogenous composition of these cytoskeletal IF proteins in childhood glial tumors may reflect a direct stage dependent correlation with their neoplastic transformation.
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PMID:Co-expression of four intermediate filament subclasses in childhood glial neoplasms. 172 88

In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoclonal antibody NE14, as early as P11, not at P3, and only in a few axons. At P18-19 and more markedly at P29, many more callosal axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as P11, but not at P3. Between P11 and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural study. These cytoskeletal changes may terminate the juvenile-labile state of callosal axons and allow further axial growth of the axon.
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PMID:Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat. 212 24

Immunophenotype analysis of 17 childhood medulloblastoma (MED) and supratentorial primitive neuroectodermal tumors (SPNET) was performed on frozen sections using 16 monoclonal antibodies (MoAb) with the biotin-streptavidin alkaline phosphatase immunohistochemical technique. Neuroectodermal associated antigens, reacting with MoAb UJ13/A, UJ127.11, UJ167.11, and UJ223.8 were detected on greater than 10% of the cells in 15 of 17 MED/SPNET. Thy-1 was present on 14 of 17 tumors and absent on two of three SPNET. Neuronal (NF) and glial (GFAP) differentiation markers were evaluated. NF-H was demonstrated in 15 of 17, NF-M in six of 17 and NF-L in one of 17 tumors; GFAP was positive in nine of 17 patients. In nine of 17 MED/SPNET both proteins were present within the same tumor. Common leukocyte antigen was demonstrated on greater than 50% of the cells in four of 14 tumors as were shared tumor/leukocyte markers using monoclonal antibodies Thy-1, PI153/3, UJ308. The most frequent MED immunophenotype analysis was UJ 13/A+, UJ 127.11+, UJ 167.11+, UJ223.8+, PI 153/3+, A2B5+, GFAP+, NF-H+, and CLA-, NF-M-, NF-L-, 215-, 275-, 282.1-. The authors conclude that MED and SPNET are heterogeneous for expression of 16 markers and have similar immunophenotype analysis profiles, supporting the concept of their common, neuroectodermal origin. Common leukocyte antigen on both tumor cells and leukocytes precludes identification of tumor infiltrating leukocytes using monostaining techniques.
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PMID:Immunophenotype profile of childhood medulloblastomas and supratentorial primitive neuroectodermal tumors using 16 monoclonal antibodies. 219 9

Effects of dephosphorylation on interactions of neurofilaments (NFs) with microtubules (MTs) were studied by the cosedimentation method. Centrifugation conditions were chosen so that MTs pelleted but NFs did not. While NFs isolated from bovine spinal cords did not cosediment with MTs polymerized in the presence of taxol, NFs dephosphorylated with Escherichia coli alkaline phosphatase began to coprecipitate with MTs. The dephosphorylated NFs bound to MTs but not to the unpolymerized tubulin dimer. The binding was not observed in the presence of high salt or with MTs containing microtubule-associated proteins. The cosedimentation experiments using purified NF subunit proteins showed that the dephosphorylation-induced binding of NFs to MTs was mediated by the largest subunit of NF (NF-H). Negative staining electron microscopy confirmed bindings of the dephosphorylated NFs and NF-H to MTs. Densitometric measurement of the bound and unbound NF-H after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the binding of the dephosphorylated NF-H to MT was saturable and gave the following binding parameters. Approximately 1 mol of NF-H bound per 10 mol of tubulin dimer with a high affinity site (Kd = 3.8 x 10(-8) M) and per 16 mol of tubulin dimer with a low affinity site (Kd = 1.1 x 10(-7) M).
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PMID:Dephosphorylation-induced interactions of neurofilaments with microtubules. 225 37

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.
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PMID:Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilament, and microtubule-associated proteins. 243 80

The phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye ("Stains-all") binding to NF polypeptides resolved on SDS-polyacrylamide gels. Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively. Incubation of NF preparations with E. coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L. This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms. "Stains-all" was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins. Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes. Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively. NF-H bound the dye less efficiently than was expected from its phosphate content. After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with "Stains-all," the staining intensity of NF-M decreased by 20-40%, and the staining of NF-L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments. 244 39

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.
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PMID:Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis. 246 77

The low (NF-L) and middle (NF-M) molecular weight (Mr) neurofilament (NF) subunits are expressed before the high (NF-H) Mr NF subunit in embryonic neurons. Thereafter, NF-M attains its mature state of phosphorylation more rapidly than does NF-H. However, little is known about NF subunit expression during cell division. A rapidly dividing medulloblastoma cell line (D283 MED), therefore, was examined using flow cytometry, immunochemistry, and a large panel of NF subunit-specific polyclonal and monoclonal antibodies. Many of the monoclonal antibodies (MAbs) distinguished NF-H and NF-M in different states of phosphorylation. By flow cytometry, more than 90% of the D283 cells expressed NF-H and NF-M in different states of phosphorylation, and an antiserum specific for the carboxy terminus of NF-L labeled more than 60% of these cells. Furthermore, the fluorescence intensity produced by MAbs that detected phosphorylated versus nonphosphorylated NF-H and/or NF-M epitopes, appropriately decreased or increased, respectively, by preincubating the D283 cells with alkaline phosphatase. In contrast, cell staining with antibodies specific for phosphate-independent NF protein epitopes did not change substantially as a result of enzymatic dephosphorylation. These results agreed closely with those obtained from studies of normal human spinal cord NF extracts. However, NF-H, NF-M, and NF-L were expressed throughout the cell cycle in dual parameter studies of D283 cells labeled with an antibody and propidium iodide. Nevertheless, reductions in the fluorescence intensity produced with most of these antibodies late in the cell cycle suggested that NF proteins may be subject to modifications in their structure or accessibility to antibody probes during different phases of the cell cycle. These data led to the conclusion that NF subunits are expressed throughout the cell cycle in cultured human medulloblastoma cells, but that subtle changes in the immunoreactivity of these proteins occur during cell division.
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PMID:Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells. 247 22

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.
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PMID:The effects of dephosphorylation on the structure of the projections of neurofilament. 253 87

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.
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PMID:Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH. 255 34


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