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

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.
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PMID:Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells. 1588 98

The objective of this study was to identify the receptor of OJ367, an oyster juice-borne bacteriophage, in Salmonella derby ATCC 6960. The crude receptor outer membrane (OM) fraction was prepared and examined from the total cell envelope (TCE) by differential extraction with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES)-MgCl2 and then with Triton-HEPES-ethylenediamine tetra-acetic acid buffers. The OM proteins (Omps) were isolated by diethylaminoethyl column chromatography to screen for receptor activity. A 45-kDa protein belonging to a minor Omp species, with phage neutralization ability, was eluted in a homogeneous form. It was a non-peptidoglycan-associated protein which was digestible by trypsin. Lipopolysaccharide had no influence on its receptor activity when coexistent in the diethylaminoethyl column fractions. An S. derby mutant resistant to lysis by phage OJ367 was isolated. The mutant not only showed decreased receptor activity in vitro when its TCE was tested but had an altered Omp profile. This implied that the 45-kDa Omp is involved as a receptor in coliphage binding; however, this role is affected by the expression of other Omps.
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PMID:The receptor of an oyster juice-borne coliphage OJ367 in the outer membrane of Salmonella derby. 1634 40

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG-Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent K(m) and V(max) of Des2 for the substrate N-octanoylsphinganine were 35 microM and 40 nmol x h(-1) x mg of protein(-1) respectively. The K(m) of the hydroxylase for mb5 was 0.8 microM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.
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PMID:Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5. 1657 Nov 4

The properties of the plasma membrane H(+)-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L., cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37 degrees C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K(+) in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This ;latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit.
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PMID:Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation. 1666 62

Microsomal membrane preparations from maize (Zea mays L., inbred A636) endosperm cultures contained enzymes that transferred sugar moieties from uridine diphosphate-N-acetylglucosamine, guanosine diphosphate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. These enzyme activities were characterized with respect to detergent and pH optima, substrate kinetic constants, and product and antibiotic inhibition constants. It was demonstrated by mild acid hydrolysis and high performance liquid chromatography that the products of the N-acetylglucosamine transferases were N-acetylglucosamine-pyrophosphoryl-dolichol and N,N'-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the product of the mannose transferase was mannosyl-phosphoryl-dolichol. A large proportion of the products of the glucose transferase activity was stable to mild acid hydrolysis. However, the proportion that was labile was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation and isopycnic banding in linear sucrose density gradients in the presence of 1 millimolar ethylenediaminetetraacetic acid indicated that the glycosyltransferase activities were located in the endoplasmic reticulum. The glycosyltransferases were not solubilized by 500 millimolar KCl or by sequential washes with tris-(hydroxymethyl)aminomethane and water, treatments that release peripheral membrane proteins. Solubilization was achieved with low concentrations of Triton X-100. When sealed microsomal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substantially reduced, while the activity of the glucosyl-transferase was somewhat reduced. Activity of the mannosyl-transferase was resistant to inactivation by incubation with trypsin unless Triton was present.
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PMID:Glycoprotein synthesis in maize endosperm cells: the nucleoside diphosphate-sugar: dolichol-phosphate glycosyltransferases. 1666 57

Previous attempts to create small-caliber vascular prostheses have been limited. The aim of this study was to generate tissue-engineered small-diameter vascular grafts using decellularized ureters (DUs). Canine ureters were decellularized using one of four different chemical agents [Triton-X 100 (Tx), deoxycholate (DCA), trypsin, or sodium dodecyl sulfate (SDS)] and the histology, residual DNA contents, and immunogenicity of the resulting DUs were compared. The mechanical properties of the DUs were evaluated in terms of water permeability, burst strength, tensile strength, and compliance. Cultured canine endothelial cells (ECs) and myofibroblasts were seeded onto DUs and evaluated histologically. Canine carotid arteries were replaced with the EC-seeded DUs (n = 4). As controls, nonseeded DUs (n = 5) and PTFE prostheses (n = 4) were also used to replace carotid arteries. The degree of decellularization and the maintenance of the matrix were best in the Tx-treated DUs. Tx-treated and DCA-treated DUs had lower remnant DNA contents and immunogenicity than the others. The burst strength of the DUs was more than 500 mmHg and the maximum tensile strength of the DUs was not different to that of native ureters. DU compliance was similar to that of native carotid artery. The cell seeding test resulted in monolayered ECs and multilayered alpha-smooth muscle actin-positive cells on the DUs. The animal implantation model showed that the EC-seeded DUs were patent for at least 6 months after the operation, whereas the nonseeded DUs and PTFE grafts become occluded within a week. These results suggest that tissue-engineered DUs may be a potential alternative conduit for bypass surgery.
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PMID:Decellularized ureter for tissue-engineered small-caliber vascular graft. 1860 13

The brain vascular endothelium operates as a dynamic regulatory interface to maintain the cell environment of the nervous system. In the vicinity of astrocytes, brain endothelial cells develop characteristic features conferring a strong cellular impermeability which limits the penetration of various compounds. The aim of our study was to determine by differential proteomic analysis the changes occurring in bovine brain capillary endothelial cells (BBCEC) differentiated in co-culture with astrocytes compared with endothelial cells cultured alone. In order to obtain reproducible and meaningful protein profiles of in vitro blood-brain barrier models, three sample preparation procedures were carried out to provide the first 2-D comparative proteomic study of BBCEC. Our study highlights advantages and drawbacks of each procedure. The cellular proteins prepared from mechanical scraping of collagen-seeded BBCEC were strongly contaminated by serum proteins. Enzymatic dissociation of BBCEC by trypsin or collagenase solved this problem. A comparative 2-DE profile study of collagenase-harvested BBCEC revealed that cytoskeleton-related proteins (actin, gelsolin and filamin-A) show the most significant quantitative changes in the Triton soluble protein fraction from BBCEC that exhibit characteristics closest to the in vivo situation.
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PMID:Actin, gelsolin and filamin-A are dynamic actors in the cytoskeleton remodelling contributing to the blood brain barrier phenotype. 1920 8

The objectives of this study were to develop a three-dimensional acellular cartilage matrix (ACM) and investigate its possibility for use as a scaffold in cartilage tissue engineering. Bovine articular cartilage was decellularized sequentially with trypsin, nuclease solution, hypotonic buffer, and Triton x 100 solution; molded with freeze-drying process; and cross-linked by ultraviolet irradiation. Histological and biochemical analysis showed that the ACM was devoid of cells and still maintained the collagen and glycosaminoglycan components of cartilage. Scanning electronic microscopy and mercury intrusion porosimetry showed that the ACM had a sponge-like structure of high porosity. The ACM scaffold had good biocompatibility with cultured rabbit bone marrow mesenchymal stem cells with no indication of cytotoxicity both in contact and in extraction assays. The cartilage defects repair in rabbit knees with the mesenchymal stem cell-ACM constructs had a significant improvement of histological scores when compared to the control groups at 6 and 12 weeks. In summary, the ACM possessed the characteristics that afford it as a potential scaffold for cartilage tissue engineering.
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PMID:Fabrication and repair of cartilage defects with a novel acellular cartilage matrix scaffold. 1989 38

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.
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PMID:Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations. 2048 38

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase. Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9-10S and 4.5-4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G(1) form.
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PMID:Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes. 2050 Nov 3


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