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)

Pretreatment by hypothermic (25 degrees C) cycling (PHC) of attached exponential-phase V79 Chinese hamster cells by Method 4 (24 hr at 25 degrees C + 1.5 hr at 37 degrees C + 24 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) or by Method 3 (48 hr at 25 degrees C + trypsin + 3 hr at 37 degrees C) make mammalian V79 cells significantly more resistant to 43 degrees C hyperthermia. There is no significant difference in the 43 degrees C curves whether Method 3 or 4 is used for pre-exposure. If pre-exposure at 15 or 10 degrees C, the resistance to hyperthermia is significantly reduced. PHC by Method 4 significantly increases survival of cells exposed to 5 degrees C and, to a lesser extent, to 10 degrees C. The increase in hyper- and hypothermic survival after PHC cannot be accounted for by changes in cell cycle distribution. Heat-shock protein synthesis is not induced by PHC; hence, protection does not result from newly synthesized proteins. When cells are made tolerant to hyperthermia by a pretreatment in 2% DMSO for 24 hr at 37 degrees C (Method 8), the cells are not more resistant to subsequent exposures to hypothermia, either at 5 or 10 degrees C. The results imply that there may be two mechanisms of inducing resistance to hyperthermia, only one of which also confers resistance to hypothermia.
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PMID:Induction of tolerance to hypothermia and hyperthermia by a common mechanism in mammalian cells. 831 51

Attached asynchronous exponential phase V79 Chinese hamster cells were pretreated by hypothermic cycling in Hepes growth medium by Method 3 (48 h at 25 degrees C + trypsin) or Method 4 (48 h at 25 degrees C + 3 h at 37 degrees C + trypsin) prior to a freeze-thaw (FT) cycle in Hepes growth medium. Pretreatment by Method 3 or 4 increased the FT survival by a factor of 3.4. This implies that the 3 h at 37 degrees C, after the 25 degrees C exposure, is not necessary to confer resistance to the subsequent FT cycle in the case of V79 cells. However, with RIF-1 mouse cells, the 3 h at 37 degrees C confers increased resistance. The increase in FT survival of V79 cells after the above pretreatments cannot be accounted for by changes in cell cycle age distribution. No heat shock proteins are produced by this pretreatment. Since pretreatment by Method 3 or 4 also makes the cells resistant to hyperthermia, three other pretreatments, making the cells thermotolerant, were tried. None of these pretreatments resulted in a change in FT survival of the cells. Interaction analysis of FT data, when pretreatment by Method 4 is combined with the presence of DMSO during the FT cycle, indicates that the pretreatment and DMSO act synergistically whether exponential or stationary phase cells are used. Furthermore, the pretreatments and L-glutamine also act synergistically. These pretreatments also increase the FT survival of the RIF-1 mouse cell line; again, pretreatment and DMSO act synergistically. Hence, the method is not limited to cells of hibernating mammals.
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PMID:Induction of tolerance to freeze-thaw (FT) damage in mammalian cells by pre-FT hypothermia treatment. 840 86

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.
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PMID:Characterization of a human basophil-like cell line (LAMA-84). 869 92

We have characterized an activity in sea urchin eggs which prevents microtubule assembly at minus ends. Using Chlamydomonas axoneme fragments to nucleate the assembly of plus and minus end microtubules, we find robust assembly at microtubule plus ends with negligible assembly at minus ends. The minus end assembly inhibitor does not co-pellet with microtubules when assembly is stimulated with DMSO while the resulting pellet of tubulin and microtubule associated proteins readily assembles from both plus and minus ends of axoneme fragments. Addition of increasing concentrations of porcine bran tubulin to the tubulin and MAP-depleted fraction eventually saturates the minus end inhibitory activity. Compared to purified tubulin, cytosolic fractions both increase the minus end critical concentration approximately 3 fold and decrease the plus end critical concentration. The inhibitory activity is removed by heating, trypsin, or by co-immunoprecipitation with tubulin. We hypothesize that a tubulin dimer binding protein is responsible for preventing assembly onto minus ends in our in vitro assays and speculate that this protein functions in vivo to prevent spontaneous nucleation, thus limiting assembly to nucleation sites.
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PMID:Mechanisms blocking microtubule minus end assembly: evidence for a tubulin dimer-binding protein. 887 19

Acipenserid fish sperm possess trypsin-like activity, resembling acrosin activity of mammalian sperm, which can be measured by hydrolysis of N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) (Ciereszko et al., 1994: J Exp Zool 268:486-491). We found that this activity can be preserved when sperm is frozen on dry ice with 0.6 M sucrose-10% dimethylsulfoxide (DMSO) extender (sperm:extender ratio 1:3), and subsequently stored in liquid nitrogen. However, other methods of freezing (without cryoprotectant at -18 degrees C, -80 degrees C, and -196 degrees C) did not protect this activity. Acrosin-like activity decreased in the course of storage of milt on ice; 88% decline was recorded after 13 days. Acrosin-like activity increased with temperature from 10 degrees C to 30 degrees C, but was inactivated at 40 degrees C to about 40% as compared to the optimum temperature. Triton X-100 inhibited activity by 15% and 72% at 0.01% and 0.1% concentrations, respectively. Activity was not affected by Mg2+ but was inhibited by Zn2+ (30% and 75% in the presence of 0.1 mM and 1 mM, respectively). Maximum velocity of substrate hydrolysis was observed at 2 mM of BAPNA. Acrosin-like activity was effectively inhibited by 4'-acetamidophenyl 4-guanidinobenzoate (AGB), an inhibitor of mammalian acrosin. Sperm acrosin-like activity correlated negatively with antiproteinase activity of seminal plasma. We conclude that sturgeon acrosin-like activity shares many properties with mammalian acrosin. On the other hand, it has some unique properties which may represent adaptations of this enzyme to the environment of external fertilization.
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PMID:Characterization of acrosin-like activity of lake sturgeon (Acipenser fulvescens) spermatozoa. 887 72

In an accompanying manuscript, it was shown that the cartilage chondrolytic activities of fibronectin fragments (Fn-f), which are mediated through catabolic cytokines such as TNF-alpha, IL-1 and IL-6, could be suppressed by anti-oxidants (AOs). The AOs neutralized reactive oxygen species (ROS) which are known to mediate catabolic cytokine action. The objective in this work was to test whether AOs would promote restoration of proteoglycan (PG) in Fn-f treated cartilage, since under normal culturing conditions, PG is not restored after removal of the Fn-f. Cartilage was first cultured with an amino-terminal 29-kDa Fn-f to cause loss of about half of the total PG and then treated with NAC (1 and 10 mM) or glutathione (10 microM) or DMSO (0.1 or 1%). Treatment with NAC and glutathione maximally caused restoration of PG within 14 days to normal or supernormal levels, while DMSO was less effective. Catalase, but not superoxide dismutase, enhanced PG content to a small but significant extent. The restoration of PG in Fn-f treated cartilage occurred throughout the full depth of the cartilage slices as shown by histochemical analysis. However, removal of the AO allowed a subsequent decrease in PG content suggesting that the AOs had not blocked cytokine expression but had merely suppressed cytokine activities. Addition of NAC to IL-1 treated cartilage promoted a restoration of PG, while addition to chymopapain or trypsin treated cartilage was not very effective, suggesting that the effect of AOs requires a cytokine driven damage system. We conclude that the AOs promote a restoration of PG in the Fn-f treated cartilage by suppressing the effects of catabolic cytokines. The data suggest a potential for AOs in reversing tissue damage caused by cytokines.
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PMID:Fibronectin fragment mediated cartilage chondrolysis. II. Reparative effects of anti-oxidants. 895 Feb

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.
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PMID:Protein purification with vapor-phase carbon dioxide. 1009 36

Cholesterol sulfate (CS) and sulfatides in the epithelium of the digestive tract were found in the 1000xg supernatants of digestive fluid, particularly in gastric juices containing the duodenal contents and bile acids, there being 14-131 microg of CS and 3-54 microg of sulfatides per mg of protein in the fluid, respectively. CS and sulfatides dissolved in detergents including bile acids inactivated pancreatic trypsin to the same level as by DMSO-solubilized sulfated lipids at 37 degrees C. Similarly, pancreatic DNase I was inhibited by CS solubilized with DMSO or bile acids, but not by sulfatides or other membrane lipids at 37 degrees C. Both the sulfate group and the hydrophobic side chain of CS were indispensable structures for the inhibition of DNase I. Also, the optimum molar ratio of bile acids to CS was important for expression of the inhibitory activity of CS toward DNase I, it being 0.18 of the optimum ratio for sodium taurocholate, and the molar ratio of CS to DNase I for complete inhibition was 342:1. Thus, CS was shown to play a role as an epithelial inhibitor of DNase I in concert with bile acids.
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PMID:Shedding of sulfated lipids into gastric fluid and inhibition of pancreatic DNase I by cholesterol sulfate in concert with bile acids. 1101 78

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.
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PMID:Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor. 1259 66

Proteomic analysis is an important approach to characterize the proteome and study protein functions. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be overlooked by conventional biochemical techniques. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an alkylating agent that can induce nontargeted mutagenesis in treated cells, although the mechanism remains unclear. Using an efficient proteomic method, we identified several cellular proteins that are responsive to low-concentration MNNG treatment in human FL cells. After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images then were analyzed with 2D analysis software. More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). Thirty-one proteins only detected in MNNG-treated or control cells were subjected to in-gel digestion with trypsin and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting. Eighteen of theses proteins have been identified, including several zinc finger proteins, two members of the ADAMs (a disintegrin and metalloprotease domain) family, and two integrins. Most of these proteins have unknown functions and their involvement in the cellular responses to alkylating agents have not been reported. Therefore, our findings may offer new insights into the mechanisms of low-concentration MNNG-induced nontargeted mutagenesis and these proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens.
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PMID:Altered expression of zinc finger proteins, ADAMs, and integrin-related proteins following treatment of cultured human cells with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine. 1280 5


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