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

---

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

Lysosomal degradation of the ganglioside GM2 by human beta-hexosaminidase A requires the presence of the GM2 activator protein as an essential cofactor. Here we demonstrate that GM2 activator mRNA is differentially expressed and mainly localized to the apical part of the epithelial cells of distal renal tubules and the collecting duct. In order to understand the mechanism underlying the regulation of the GM2 activator gene, we analyzed the genomic organization upstream exon 2 as well as the 5'-flanking region. The GM2 activator gene spans about 16.8 kb with a first intron of 6.5 kb, and the transcription start is located at position -96 upstream from the ATG. DNA elements responsible for GM2 activator expression were identified in a PCR-based method of long-distance DNA walking. Sequence analysis revealed a 2.9 kb region upstream of the ATG that contained regulatory elements like CAAT boxes, Sp1 binding sites as well as AP1, and AP2 sites. Transfection experiments in COS-1 cells with a series of chimeras of 5'-stepwise deletion mutants of the GM2 activator gene 5'-flanking region and the secretory alkaline phosphatase (SEAP)-reporter gene indicated that a genomic fragment encompassing -323 to +1 bp had significant promoter activity. EMSA experiments showed that Sp1 and other transcription factors like AP1, AP2 and CCAAT-Box binding proteins are involved in GM2 activator gene regulation.
...
PMID:Characterization of regulatory elements in the 5'-flanking region of the GM2 activator gene. 1098 59

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.
...
PMID:Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system. 1098 47

Five novel antibiotics described as irpexans (1, 2, 3a, 3b, 4) were isolated from fermentations of an Irpex species in the course of a screening for new inhibitors of AP-1 and NF-kappaB mediated signal transduction pathways in COS-7 cells using secreted alkaline phosphatase (SEAP) as a reporter gene. The expression of an AP-1 and NF-kappaB driven SEAP reporter gene was inhibited in a dose dependent manner with 14-acetoxy-15-hydroxyirpexan (3b) being the most potent compound, followed by 14,15-irpexanoxide (2), 14,15-dihydroxyirpexan (3a) and 14-acetoxy-22,23-dihydro-15,23-dihydroxyirpexan (4). Irpexan (1) exhibited no activity. The irpexans (1, 2, 3a, 3b, 4) are characterized by weak cytotoxic but neither antibacterial nor antifungal activities. All five compounds are terpenoids with a mannose moiety. The structures were elucidated by spectroscopic methods.
...
PMID:The irpexans, a new group of biologically active metabolites produced by the basidiomycete Irpex sp. 93028. 1113 59

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. The disease is highly variable in its clinical expression, because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was found, despite exhaustive sequencing of the gene, suggesting that missing mutations are harbored in intron or regulatory sequences or that the disease is dominantly transmitted. The distinction between these two situations is of importance, especially in terms of genetic counseling, but dominance is sometimes difficult to conclusively determine by using familial analysis since expression of the disease may be highly variable, with parents of even severely affected children showing no or extremely mild symptoms of the disease. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, I473F) found in patients with no other detectable mutation. Three of these mutations, G46 V, S164L, and I473F, have not previously been described. Pedigree and/or serum alkaline phosphatase data suggested possible dominant transmission in families with A99T, R167 W, and G232 V. By means of site-directed mutagenesis, transfections in COS-1 cells, and three-dimensional (3D) modeling, we evaluated the possible dominant effect of these eight mutations. The results showed that four of these mutations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effect by inhibiting the enzymatic activity of the heterodimer, whereas the four others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of the disease. Analysis of the 3D model of the enzyme showed that mutations exhibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular biological function such as dimerization, tetramerization, or membrane anchoring.
...
PMID:A molecular approach to dominance in hypophosphatasia. 1147 41

The bone morphogenic proteins (BMPs) play a key role in skeletal development and patterning. Using the technique of differential display polymerase chain reaction (ddPCR), we have identified a novel gene whose expression is increased during BMP-2-induced differentiation of the prechondroblastic cell line, MLB13MYC clone 17, to an osteoblastic phenotype. The 6.5-kilobase mRNA recognized by this ddPCR product is increased 10-fold by BMP-2 treatment of the MLB13MYC clone 17 cells. The mRNA recognized by this ddPCR product is also increased as MC3T3-E1 cells recapitulate the program of osteoblast differentiation during prolonged culture. The full-length transcript corresponding to this ddPCR product was cloned from a MLB13MYC clone 17 cell cDNA library. Analysis of the deduced amino acid sequence demonstrated that this gene encodes a novel 126-kDa putative serine/threonine protein kinase containing a nuclear localization signal. The kinase domain, expressed in Escherichia coli, is capable of autophosphorylation as well as phosphorylation of myelin basic protein. The gene was, therefore, named BIKe (BMP-2-Inducible Kinase). The BIKe nuclear localization signal is able to direct green fluorescent protein to the nucleus in transfected COS-7 cells. When stably expressed in MC3T3-E1 cells, BIKe significantly decreases alkaline phosphatase activity and osteocalcin mRNA levels and retards mineral deposition relative to vector control. This novel kinase, therefore, is likely to play an important regulatory role in attenuating the program of osteoblast differentiation.
...
PMID:Cloning and characterization of a novel protein kinase that impairs osteoblast differentiation in vitro. 1150 May 15

Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.
...
PMID:Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153-->Asp substitution, a cause of perinatal hypophosphatasia. 1180 76

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.
...
PMID:Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165. 1188 73

CNS myelin contains axon outgrowth inhibitors, such as Nogo, that restrict regenerative growth after injury. An understanding of the mechanism of Nogo signaling through its receptor (NgR) is critical to developing strategies for overcoming Nogo-mediated inhibition. Here we analyze the function of NgR domains in outgrowth inhibition. Analysis of alkaline phosphatase (AP)-Nogo binding in COS-7 cells reveals that the leucine-rich repeat domain is necessary and sufficient for Nogo binding and NgR multimerization. Viral infection of embryonic day 7 chick retinal ganglion cells with mutated NgR demonstrates that the NgR C-terminal domain is required for inhibitory signaling but not ligand binding. The NgR glycosylphosphatidylinositol domain is not essential for inhibitory signaling but may facilitate Nogo responses. From this analysis, we have developed a soluble, truncated version of the Nogo receptor that antagonizes outgrowth inhibition on both myelin and Nogo substrates. These data suggest that NgR mediates a significant fraction of myelin inhibition of axon outgrowth.
...
PMID:Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin. 1238 94

Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.
...
PMID:Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop. 1249 79

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.
...
PMID:Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta. 1258 78


<< Previous 1 2 3 4 5 6 7 8 9 Next >>

---