Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified a Drosophila homolog of vertebrate classic cadherins. A monoclonal antibody to Drosophila alpha-catenin (D alpha-catenin) copurifies a 150-kDa glycoprotein (gp150) along with the alpha-catenin. To further characterize this protein, we generated monoclonal antibodies to gp150 and isolated its cDNAs using the antibodies. Predicted sequences of the encoded product revealed that it is a transmembrane protein with similarity to vertebrate classic cadherins, and so we designated this molecule DE-cadherin. The extracellular domain has six cadherin-specific repeats, although the first repeat seems to be cleaved off upon maturation, and the cytoplasmic domain shows significant identity to that of vertebrate classic cadherins. DE-cadherin is distinguishable from its vertebrate counterparts by a large insertion with local sequence similarity to Fat, laminin A chain, Slit, and neurexin I at the proximal region of the extracellular domain. Despite such differences, DE-cadherin is functionally similar to vertebrate classic cadherins. For example, it is associated with alpha-catenin and beta-catenin (Armadillo), and protected from trypsin digestion only in the presence of Ca2+, as is the case for many of classic cadherins. Transfection of S2 cells with the DE-cadherin cDNA enhances their Ca(2+)-dependent cell aggregation. Antibodies to this molecule inhibited aggregation of not only the transfectants but also early embryonic cells. DE-cadherin is concentrated at the apical poles of epithelial cell-cell junctions. All these results suggest that DE-cadherin is a homolog of vertebrate classic cadherins and that the vertebrate and invertebrate share common mechanisms for regulation of cell-cell adhesion.
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PMID:A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. 795 32

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.
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PMID:M-cadherin-mediated cell adhesion and complex formation with the catenins in myogenic mouse cells. 916 9

Cloning of rat cadherin-8 cDNA demonstrated two types of cDNAs. The overall structure of the protein defined by one type of the cDNA is essentially the same as that of classic cadherins, whereas the protein defined by the other type of cDNA ends near the N-terminus of the fifth repeat of the extracellular domain (EC5) and contains a short unique sequence at the C-terminus. The same truncated type of cDNA was also obtained from a human cDNA library. In Northern blot analysis of rat brain mRNA, a probe for EC5 detected multiple bands of about 3.5-4.3 knt, whereas a probe for the alternative form hybridized with a band of about 3.5 knt. Western blot experiments showed that an antibody against the extracellular domain of rat cadherin-8 stained a band of about 95 kDa and a faint band of about 130 kDa in rat brain extract. These results suggest that cadherin-8 is expressed in two forms, a complete form and a truncated form without a transmembrane domain or cytoplasmic domain, in brain. The complete form of cadherin-8 expressed in L cells was about 130 kDa in molecular mass and was located at the cell periphery, mainly at the cell-cell contact sites. However, we failed to express the truncated form in L cells. The transfectants of the complete form showed weak cell adhesion activity. The complete form of cadherin-8 was sensitive to trypsin digestion, and Ca2+ did not protect cadherin-8 from digestion, in contrast to the classic cadherins. The complete form of cadherin-8 coprecipitated with beta-catenin, but did not immunoprecipitate well with alpha-catenin or gamma-catenin. Cadherin-8, as well as cadherin-11, was mapped to a specific region of chromosome 8 that also includes cadherins-1, -3, and -5.
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PMID:Molecular properties and chromosomal location of cadherin-8. 952 72

We used a novel cDNA cloning method based on the cadherin-beta-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca2+ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.
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PMID:Molecular cloning and characterization of a novel human classic cadherin homologous with mouse muscle cadherin. 954 47

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.
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PMID:Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets. 1082 21

Cell adhesion receptors of the cadherin family are involved in various developmental processes, affecting cell adhesion and migration, and also cell proliferation and differentiation. In order to dissect the molecular mechanisms of cadherin-based cell-cell adhesion and subsequent signal transduction to the cytoskeleton and/or cytoplasm leading to adapted cell responses, we developed an approach allowing us to mimic and control cadherin activation. We produced a dimeric N-cadherin-Fc chimera (Ncad-Fc) which retains structural and functional properties of cadherins, including glycosylation, Ca(2+)-dependent trypsin sensitivity and the ability to mediate Ca(2+)-dependent self-aggregation of covered microbeads. Beads covered with either Ncad-Fc or anti-N-cadherin antibodies specifically bound to N-cadherin expressing cells. Both types of beads induced the recruitment of N-cadherin, beta-catenin, alpha-catenin and p120, by lateral mobilization of preexisting cell membrane complexes. Furthermore, cadherin clustering elicited by Ncad-Fc beads triggered local accumulations of tyrosine phosphorylated proteins, a recruitment and redistribution of actin filaments, as well as local membrane remodeling. These results support a model where the adhesion of cadherin ectodomains is followed by clustering of cadherin/catenin complexes allowing signal transduction affecting both cytoskeletal reorganization and cytoplasmic signal mobilization (outside-in signaling). Interestingly, bead-cell binding was altered by agents promoting microfilament and microtubule depolymerization or tyrosine phosphorylation, indicating a possible regulation of the adhesive properties of the extracellular domain of N-cadherin by intracellular factors (inside-out signaling).
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PMID:Immobilized dimers of N-cadherin-Fc chimera mimic cadherin-mediated cell contact formation: contribution of both outside-in and inside-out signals. 1082 93

The adenomatous polyposis coli (APC) tumor suppressor protein is involved in the Wnt/wingless pathway, modulating beta-catenin activity. We report the development of a highly specific, chemically synthesized oligobody (oligonucleotide-based synthetic antibody), directed against the N-terminal region of APC. Using this reagent, we found that within 16 h of disrupting HT-29 cell-cell contacts by harvesting cells with trypsin/EDTA treatment and replating, APC was translocated from the cytoplasm to the nucleus. Five days after plating the cells, when the cells had returned to their normal confluent phenotype and cell-cell contacts were reestablished, APC returned to the cytoplasm. These results suggest that APC functions as part of a "sensor" system, and responds to the loss of cell-cell contacts by moving to the nucleus, and returning to the cytoplasm when the contacts are fully restored.
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PMID:APC senses cell-cell contacts and moves to the nucleus upon their disruption. 1140 91

The native three-dimensional architecture of carcinomas, which governs numerous autocrine-paracrine interactions related to tumor progression, cannot be faithfully recreated in most in vitro models. Even when the three-dimensional architecture is recreated in artificial scaffolds such as soft agar, this approach does not truly recreate the natural microenvironment of the tumor. Multicellular spheroids can reasonably recreate in vitro the natural three-dimensional architecture of carcinomas, but even the most efficient gene delivery vectors will penetrate only the outer layers of these structures and hence only a small fraction of cells will receive the gene of interest. If the multicellular spheroids are disrupted into a single-cell suspension in order to achieve high transfection efficiency, the single-cell production may have so altered the gene expression profile of the spheroid as to bring into question its present relevancy to in vivo tumor progression. Our laboratory has developed a human-SCID (severe combined immunodeficient) mouse model of inflammatory breast cancer, MARY-X, which grows as tight multicellular spheroids in vitro and as lymphovascular emboli in vivo. The spheroids, which express only low levels of surface sialyl-Lewis(x/a) (sLe(x/a)), are able to form compact homotypic cell clumps mediated by an intact, overexpressed E-cadherin/alpha,beta-catenin axis. The spheroids can be fully disrupted by trypsin proteolysis, anti-E-cadherin antibodies, or Ca(2+) depletion. Of these approaches the disruption with depleted Ca(2+), complete after 30 min, is fully reversible by the readdition of Ca(2+) within 6 hr. This time interval allows for a transfection "window" in which successful gene delivery can be achieved before spheroid reformation. Retroviruses (10(6)-10(7) CFU/ml) carrying the gene encoding either green fluorescent protein (GFP), a dominant-negative E-cadherin mutant (H-2K(d)-E-cad), its control (H-2K(d)-E-cad Delta C25), or alpha-1,3-fucosyltransferase III (FucT-III), an enzyme that increases surface sLe(x/a), were used to transfect either intact (wild-type) or disadhered/readhered (reformed) spheroids. There were marked differences in transfection efficiency in the reformed versus wild-type spheroids. Retroviral transfection of GFP resulted in successful delivery of this reporter gene to only the outer layer of cells of the wild-type spheroids, but to all layers of the reformed spheroids. A single retroviral transfection of H-2K(d)-E-cad, H-2K(d)-E-cad Delta C25, or FucT-III produced evidence of their respective gene expression at 72 hr throughout all layers of the reformed spheroids, but only H-2K(d)-E-cad and FucT-III produced progressive disadherence. Both H-2K(d)-E-cad-MARY-X and FucT-III-MARY-X lost their ability to form lymphovascular emboli in SCID mice. This reversible model of spheroid formation has provided us with insight into the pathogenesis of inflammatory breast carcinoma. If more broadly applied, this model could be used to examine the effects of any gene, using any gene delivery system in the three-dimensional context of native tumoral architecture.
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PMID:Reversible model of spheroid formation allows for high efficiency of gene delivery ex vivo and accurate gene assessment in vivo. 1213 77

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.
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PMID:In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body. 1591 77

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene seem to underlie the initiation of many colorectal carcinomas. Loss of APC function results in accumulation of beta-catenin and activation of beta-catenin/TCF-dependent transcription. Recent studies have implicated APC in controlling retinoic acid biosynthesis during normal intestinal development through a WNT-independent mechanism. Paradoxically, however, previous studies found that dietary supplementation of Apc(MIN) mice with retinoic acid failed to abrogate adenoma formation. While investigating the above finding, we found that expression of CYP26A1, a major retinoic acid catabolic enzyme, was up-regulated in Apc(MIN) mouse adenomas, human FAP adenomas, human sporadic colon carcinomas, and in the intestine of apc(mcr) mutant zebrafish embryos. Mechanistically, cyp26a1 induction following apc mutation is dependent on WNT signaling as antisense morpholino knockdown of tcf4 or injection of a dnLEF construct into apc(mcr) mutant zebrafish suppressed expression of cyp26a1 along with known WNT target genes. In addition, injection of stabilized beta-catenin or dnGSK3beta into wild-type embryos induced cyp26a1 expression. Genetic knockdown or pharmacologic inhibition of cyp26a1 in apc(mcr) mutant zebrafish embryos rescued gut differentiation defects such as expression of intestinal fatty acid-binding protein and pancreatic trypsin. These findings support a novel role for APC in balancing retinoic acid biosynthesis and catabolism through WNT-independent and WNT-dependent mechanisms.
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PMID:Up-regulation of CYP26A1 in adenomatous polyposis coli-deficient vertebrates via a WNT-dependent mechanism: implications for intestinal cell differentiation and colon tumor development. 1688 56


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