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)

Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.
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PMID:Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins. 1822 23

Signal peptides (SP) and transmembrane segments (TMS) ensure proper subcellular targeting and localization of proteins. Thus, understanding the molecular variability of this targeting information is essential. In this study, we functionally analyzed the predicted SP and the TMS of adherens junction protein, shrew-1 (Bharti et al. Novel membrane protein shrew-1 targets to cadherin-mediated junctions in polarized epithelial cells. Mol Biol Cell 2004:15:397). We used human secreted alkaline phosphatase (SEAP) as reporter protein. The SP of shrew-1 was able to functionally substitute for SEAP's intrinsic SP and was cleaved, indicating that it acts as a start-transfer signal and not a signal anchor. In turn, the TMS of shrew-1 functions as stop-transfer signal. Notably, clearly detectable plasma membrane localization is only achieved when the fusion protein contains both the SP and the TMS of shrew-1. In combination with the intrinsic SP from SEAP, the shrew-1 TMS is unable to promote stable plasma membrane localization. Hence, it may be assumed that this synergism between an SP and a TMS to mediate plasma membrane localization is essential for structural and/or functional integrity of shrew-1.
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PMID:Synergism of shrew-1's signal peptide and transmembrane segment required for plasma membrane localization. 1848 53

Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified. Other Cry4Ba binding proteins identified include the putative lipid raft proteins flotillin and prohibitin, V-ATPase B subunit and actin. These identified proteins might play important roles in mediating the toxicity of Cry4Ba due to their location in the gut brush border membrane. Cadherin-type protein was not identified, although previously, we identified a midgut cadherin AgCad1 as a putative Cry4Ba receptor in Anopheles gambiae mosquito larvae [Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101-5110]. Other identified proteins in this study that might have lesser roles include mitochondrial proteins such as ATP synthase subunits, mitochondrial processing peptidase and porin; which are likely contaminants from mitochondria and are not brush border membrane components. Trypsin-like serine protease was also identified as a protein that binds Cry4Ba. Identification of these toxin-binding proteins will lead to a better understanding of the mode of action of this toxin in mosquito.
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PMID:Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) larvae. 1927 30

The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.
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PMID:Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. 1936 28

This study has investigated the effects of a bio-inspired ceramic surface modified with a novel recombinant protein on surface parameters and cell behavior. The surface of a biphasic calcium phosphate ceramic was functionalized with a recombinant protein spanning the fragments of fibronectin module III7-10 and extracellular domains 1 and 2 of cadherin 11 (rFN/CDH) using a dimethyl-3,3'-dithiobispropionimidate cross-linking method. The surface was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and protein adsorption and surface density measurements. The material exhibited desirable properties for cell adhesion and proliferation. The effects of the surface on the adhesion and proliferation of human mesenchymal stem cells (hMSC) were investigated using a cell adhesion centrifugal assay and the 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The data demonstrated that the adhesive capacity and proliferation rate were significantly improved as compared with fibronectin and cadherin positive controls. Moreover, the rFN/CDH bio-inspired ceramic surface also induced osteoblastic differentiation, as evidenced by the higher alkaline phosphatase activity and osteocalcin mRNA expression level of hMSC cultured in osteogenic media for 7-10days. Furthermore, a functional blocking assay with a site-specific antibody against phosphotyrosine 397 (pY397) of focal adhesion kinase revealed that pY397 is involved in adhesion and ossification. These results suggest that the rFN/CDH bio-inspired BCP surface possesses enhanced functionality in adhesion, proliferation and ossification and may be a promising scaffold for tissue engineering.
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PMID:Fabrication and characterization of a recombinant fibronectin/cadherin bio-inspired ceramic surface and its influence on adhesion and ossification in vitro. 1970 96

Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of larval BBMVs (brush border membrane vesicles). The antibody inhibits Cry11Aa toxin binding to BBMVs and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the full-length Aedes cadherin cDNA was isolated from Aedes larvae and partial overlapping fragments that covered the entire protein were expressed in Escherichia coli. Using toxin overlay assays, we showed that one cadherin fragment, which contains CR7-11 (cadherin repeats 7-11), bound Cry11Aa and this binding was primarily through toxin domain II loops alpha8 and 2. Cadherin repeats CR8-11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent Kd of 16.7 nM. Finally we showed that this Cry11Aa-binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that Aedes cadherin is possibly a receptor for Cry11A and, together with its ability to bind an ALP, suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects.
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PMID:Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. 1973 34

The limitations of specific adhesion and osteoblastic differentiation are current problems in bone tissue engineering. The aim of this study was to investigate the effect of a novel recombinant protein of fibronectin module III7-10/cadherin 11 EC1-2 (rFN/CDH) on cell adhesion and differentiation. Gene coding rFN/CDH was engineered by a homology modeling strategy, and an expression plasmid was constructed by standard DNA techniques. The rFN/CDH protein was expressed in Rosetta-gami (DE3), an improved Escherichia coli system. MC3T3-E1 cell centrifugal adhesive assay indicated that the adhesive capacity of rFN/CDH was significantly improved. Quantitative analysis of two osteogenic markers, osteocalcin mRNA expression and alkaline phosphatase activity, indicated that they were further up-regulated when human mesenchymal stem cells were cultured for 7-10 d on rFN/CDH pre-coated surfaces. These results suggest that rFN/CDH possesses an enhanced dual biofunctionality in osteoblastic adhesion and differentiation, and a promising application can be expected in biomimetic strategies and biomaterial development.
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PMID:Effect of a novel recombinant protein of fibronectinIII7-10/cadherin 11 EC1-2 on osteoblastic adhesion and differentiation. 1973 74

Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K(d) = 0.6 nM), whereas the monomer showed weaker binding (apparent K(d) = 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in beta-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.
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PMID:Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin. 2017 63

In our previous studies, roles of gap junction and vascular endothelial growth factor in the cross-talking of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) have been extensively studied. The present study focused on the investigation of the roles of neural (N)-cadherin in early differentiation of HBMSCs in direct-contact cocultures with HUVECs for 24 and 48 h. Quantitative real-time polymerase chain reaction, immunofluorescence, Western blot, as well as functional studies were applied to perform the studies at both protein and gene levels. Results showed that cocultured cells expressed much higher N-cadherin than monocultured cells after 24 and 48 h of culture. We observed that N-cadherin concentrated in the membrane of cocultured HBMSCs (co-HBMSCs) while distributed within the cytoplasm of monocultured HBMSCs, which indicated that the cell-cell adhesion was improved between cocultured cells. In addition, more beta-catenin was found to translocate into the cocultured cells nuclei and more T cell factor-1 (TCF-1) were detected in cocultured cells than in the monocultured cells. Moreover, mRNA levels of early osteoblastic markers including alkaline phosphatase (ALP) and type I collagen (Col-I) of co-HBMSCs were significantly upregulated, whereas neutralization of N-cadherin led to a downregulation of ALP and Col-I in both of the HBMSCs and co-HBMSCs compared with untreated cells. Taking our findings together it can be concluded that cocultures of HBMSCs with HUVECs increased N-cadherin expression and improved cell-cell adhesion. Whether this applies only to osteoprogenitor cells or to all the cell types in the culture will need to be determined by further studies. Subsequently, signaling transduction might be induced with the participation of beta-catenin and TCF-1. With the N-cadherin-mediated cell-cell adhesion and signaling transductions, the early osteoblastic differentiation of co-HBMSCs was significantly upregulated.
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PMID:Role of neural-cadherin in early osteoblastic differentiation of human bone marrow stromal cells cocultured with human umbilical vein endothelial cells. 2066 68

Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.
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PMID:Pore formation by Cry toxins. 2068 86


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