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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antisense oligonucleotides have therapeutic potential as inhibitors of gene expression. However, the mechanism by which an intact oligonucleotide reaches the intracellular site of action is unknown. In this study, we use an oligodeoxyribonucleotide 21-mer complementary to the translation initiation codon of the c-myc protooncogene to study the mechanism of oligonucleotide uptake and internalization into Rauscher Red 5-1.5 cells. We find trypsin-sensitive and trypsin-insensitive surface binding, in addition to internalization. Uptake is partially energy dependent and inhibited by charged molecules, including DNA, ATP, a random sequence oligonucleotide, and dextran sulfate. Uptake does not appear to occur via a traditional receptor-mediated uptake pathway because chloroquine, monensin, and phenylarsine oxide pretreatment does not significantly decrease internalization. An anion channel inhibitor, SITS, and the salts, NaCl, Na2SO4, and NH4Cl, significantly decrease oligonucleotide uptake. Whether uptake occurs via a channel or a novel uptake mechanism is still unknown. A model is proposed which reasonably simulates the experimental data.
...
PMID:Antisense c-myc oligodeoxyribonucleotide cellular uptake. 138 28

The biosynthesis of the membrane-bound metalloendopeptidase meprin was studied after the introduction of cDNAs encoding the rat meprin alpha and beta subunits into human 293 cells. When expressed individually the meprin alpha subunit was found to be primarily secreted into the culture medium, while the beta subunit was determined to be an integral membrane protein. Coexpression of the alpha and beta subunits results in the localization of both subunits to the plasma membrane. In this case the alpha subunit is specifically released from the cell surface by dithiothreitol, indicating the alpha subunit is associated with the membrane via disulfide bond(s) to the beta subunit. Meprin expressed in 293 cells is similar to the rat kidney enzyme in that it forms disulfide-linked dimers and has a similar pattern of glycosylation. However, the expressed protein displayed no detectable peptidase activity against four meprin substrates. Processing of the alpha subunit was followed after the introduction of sequences coding for the human c-myc peptide epitope EQKLISEEDL into its cDNA in the region of its prosequence and at the COOH terminus. Detection of the resulting proteins using a monoclonal antibody specific for the c-myc epitope indicates the alpha subunit is processed at its COOH terminus but retains the prosequence which is absent from the enzyme purified from rat kidney. Limited trypsin digestion of meprin precursors expressed in 293 cells results in both the activation of the enzyme and the removal of the prosequence. This result supports the hypothesis of Bode et al. (Bode W., Gomis-Ruth, F. X., Huber, R., Zwilling, R., and Stocker, W. (1992) Nature 358, 164-167) that meprin and other astacin family proteases require removal of NH2-terminal prosequences at the junction of the astacin protease domain for zymogen activation. Trypsin-activated meprin was assayed with the protein substrate azocasein and three synthetic peptide substrates. The membrane-bound beta subunit was found to be more active than the secreted alpha subunit against azocasein but much less active toward the synthetic peptide substrates.
...
PMID:Expression of meprin subunit precursors. Membrane anchoring through the beta subunit and mechanism of zymogen activation. 751 Feb 89

Flatfish leukocytes were transfected with the expression plasmids of the v-myc, c-myc, c-fos, v-myb and c-Ha-ras oncogenes. Only cotransfection of c-Ha-ras with c-myc or c-fos resulted in complete immortalization of the cells. Interferon-like anti-viral protein was found in the cultured medium of the immortalized lymphocytes. The protein was purified by DEAE-Toyopearl 650 M ion exchange chromatography and WGA agarose affinity chromatography. The protein was a glycoprotein of about 16 kDa. The antiviral activity of the protein was trypsin-sensitive and was fairly stable at pH values from 4 to 8. The protein retained about 60% of the activity even at 60 degrees C and showed a broad antiviral activity for various fish cells and viruses.
...
PMID:Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes. 768 26

Because a major limitation of ODN (oligodeoxynucleotide) use is inefficient cellular uptake, methods to improve ODN uptake could have important implications in the investigational and possibly therapeutic use of ODNs. In this study, antisense c-myc ODN cellular uptake in elevated extracellular calcium was increased up to 48-fold in the four cell lines examined. The role of calcium in ODN cellular uptake was examined using a 21-base ODN complementary to the c-myc proto-oncogene and the Rauscher cells. Cells were pretreated with uptake inhibitors in either 1.8 (physiologic) or 5.4 mM calcium prior to addition of (32P) labelled ODN. In physiologic calcium conditions, ODN cellular uptake was partially dependent on cellular energy and a trypsin-sensitive surface protein. In contrast, in the presence of elevated (5.4 mM) extracellular calcium, trypsinization and metabolic inhibition had a reduced and no effect, respectively, on uptake. Endocytosis and lysosomotropic inhibitors did not decrease uptake in either calcium concentrations. Therefore, the mechanism of ODN uptake may depend on the level of extracellular calcium. Furthermore, surface binding accounted for approximately 60% of total uptake in both physiologic and elevated calcium concentrations, suggesting that the increased uptake was not due exclusively to increased surface binding. Thus, the predominant mechanism of ODN uptake may depend on the extracellular calcium concentration.
...
PMID:Calcium dependent cellular uptake of a c-myc antisense oligonucleotide. 781 92

Previously described cell membrane transport mechanisms are unable to account completely for oligodeoxynucleotide cellular uptake. These charged macromolecules enter cells by an incompletely defined mechanism and downregulate gene expression in either the cytoplasm or nucleus. Thus, the goal of this research was to study the mechanism of phosphodiester oligonucleotide cellular uptake in Rauscher Red 5-1.5 erythroleukemia cells. An antisense c-myc oligodeoxynucleotide (21 bases) demonstrated biological activity in these cells using two types of proliferation assays and Northern blot analysis, and was internalized as visualized by confocal laser microscopy. Oligonucleotide uptake appeared to be a complex process consisting of surface binding and internalization. Cellular internalization accounted for up to 40% of total uptake and was partially dependent on both a trypsin-sensitive component and cellular energy. Uptake in these cells was nonspecific and did not appear to be due to receptor-mediated endocytosis. Therefore, because oligonucleotide cellular uptake in other cell types apparently involves an endocytic mechanism, the primary mechanism of oligonucleotide internalization may be cell line dependent.
...
PMID:Antisense c-myc oligonucleotide cellular uptake and activity. 784 86

We recently reported (1991, Mol. Cell Biol. 11, 3699-3710) that depletion of c-myc protein by myc antisense sequences in ras-transformed NIH-3T3 cells reverses several of the malignant characteristics of these cells. These include transformed morphology, growth in soft agar, and ability to form tumors in athymic mice. In the present study we examined the same cells for in vitro adhesive behavior. Cells depleted of c-myc protein by antisense transfection showed no change in attachment to fibronectin-coated dishes as compared to ras-transformed NIH-3T3 cells but had greatly increased resistance to trypsin/EDTA-mediated release from the substratum after attachment. In concomitant studies, the cells were examined for fibronectin biosynthesis and cell surface fibronectin. There was no overall change in fibronectin biosynthesis in the c-myc antisense transfected cells as compared to the ras-transformed NIH-3T3. However, immunofluorescence staining revealed increased amount of surface fibronectin associated with the antisense c-myc-expressing transfectants. Taken together, these data indicate that the conditional reacquisition of the nonmalignant phenotype in ras-transformed NIH-3T3 cells by selected depletion of c-myc protein is associated with an increase in cell-substrate adhesion. This, in turn, is associated with an increase in surface fibronectin.
...
PMID:Increased cell-substrate adhesion accompanies conditional reversion to the normal phenotype in ras-oncogene-transformed NIH-3T3 cells. 792 39

Pur alpha has been identified as a single-stranded DNA binding protein that specifically binds to the purine-rich strand present in the DNA replication initiation zone of the human c-myc gene. We have previously demonstrated that chronic morphine treatment decreases the DNA binding activity of ssCRE-BP (single-stranded cyclic AMP response element-binding protein), which has been shown to be identical to pur alpha by cDNA cloning, and is abundant in the brain. In this report we identified an activator of ssCRE-BP/pur alpha in the brain and characterized it. Although purified ssCRE-BP/pur alpha or its GST-fusion protein exhibited very low DNA binding activities, they were markedly enhanced by including nuclear extract in the binding assay. The enhanced binding activity is trypsin-sensitive, heat-stable and has a molecular weight of approximately 66 kDa. Casein could substitute for the activator and increased the DNA binding activity of ssCRE-BP/pur alpha by one order. A series of deletion mutants were prepared in order to determine the DNA binding and activator interacting domains, and both of them were found to reside in AA 50-215 of ssCRE-BP/pur alpha. These data suggest that the DNA binding activity of ssCRE-BP/pur alpha is augmented by a nuclear protein, which may modulate the ssCRE-BP/pur alpha activity to develop morphine dependence and tolerance.
...
PMID:Characterization of a nuclear factor that enhances DNA binding activity of SSCRE-BP/PUR alpha, a single-stranded DNA binding protein. 918 64

Normal human term cytotrophoblast cells prepared by trypsin-DNAse I digestion with and without secondary immunological purification with CD9 antibodies were investigated for the expression of morphological and genetic markers of proliferation and differentiation. After 24 h of culture, the cell preparations demonstrated spontaneous formation of microvilli and formation of small syncytial units as assessed by desmoplakin staining and FITC-dextran microinjection. EGF was required for mature syncytial formation. Compared to log-phase proliferating HeLa cells, uptake of [3H]thymidine incorporation was low and quickly decreased to negligible levels. Expression of the proto-oncogenes c-myc, c-fos, and c-jun and histone 2A decreased rapidly in the first 24 h of culture in both cell preparations, followed by an increase in expression of c-fos and junB over the next 3 days of culture. Proto-oncogene changes were similar in attached and suspension cells. Spontaneous increases in alpha hCG, pregnancy-specific beta(1)-glycoprotein and 3 beta-hydroxysteroid dehydrogenase (3 beta OHSD) occurred within 1 day in both cell preparations. EGF receptor blocking antibodies did not inhibit minor degrees of spontaneous syncytial formation nor inhibit spontaneous expression of alpha hCG or 3 beta OHSD mRNA, but did prevent extensive synctialization induced by EGF. The results demonstrate that term cytotrophoblast cells even in serum-free conditions or suspension culture rapidly commit to a non-proliferative differentiation program in culture which includes limited syncytialization and marked hormone mRNA expression. However, EGF is required for extensive syncytial development.
...
PMID:In vitro cultured human term cytotrophoblast: a model for normal primary epithelial cells demonstrating a spontaneous differentiation programme that requires EGF for extensive development of syncytium. 929 Jan 54

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.
...
PMID:Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines. 953 Dec 85

A new protein with nerve growth promoting activity was purified from the crude venom of the Agkistrodon halys Pallas, a Chinese snake. Its amino-terminal sequence unexpectedly showed high homology with serine proteases, suggesting that it is a new member of the serine protease family. It also cross-reacted with antibodies against thrombin-like enzyme and possessed weak arginine esterase activity, amounting to about 3% of the activity of trypsin. However, its nerve growth promoting activity was comparable to that of nerve growth factor (NGF). It was named NGF-like protease (NLP). Northern blot analysis further demonstrated different patterns of induction of c-myc, vgf and trkA mRNA transcription in PC12 pheochromocytoma cells treated with NGF and NLP, respectively. These data suggested that NLP represents a novel potent neurotrophic factor.
...
PMID:Identification of a serine protease with nerve growth promoting activity from snake venom. 985 63


1 2 Next >>

---