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)

Cell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM). Cell dispersion was performed overnight at 4 degrees C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue. Cells were examined before and after fractionation on a discontinuous low-density bovine serum albumin (BSA) gradient. Forward and right-angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure. Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells. DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy. Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells. The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.
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PMID:Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry. 241 57

It was shown that under definite conditions there was competition between natural variants of sea bacteria belonging to V. fischeri. Natural variants of V. fischeri, strain 6 differed in their resistance to streptomycin and had different growth rates under conditions of limited aeration. Morphologically all the variants were identical. V. fischeri P-0, V. fischeri P-1 and V. fischeri P-2 were studied. The study revealed that V. fischeri P-0 produced a non-dialysing thermostable trypsin-sensitive substance inhibiting the growth of V. fischeri P-1 and V. fischeri P-2. The maximum activity of the antibacterial substance was observed when V. fischeri P-0 was grown in a liquid medium with peptone and yeast extract without agitation at 26 degrees C. The inhibiting substance was also active against V. fischeri BKM B995 and V. fischeri P-7 isolated as a result of V. fischeri P-0 exposure to ethidium bromide. The substance had no effect on the following bacterial species: Aeromonas liquefaciens 301, Achromobacter liquefaciens, Pseudomonas putida 15, Pseudomonas fluorescence 7, Escherichia coli AH-32 and Staphylococcus aureus.
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PMID:[Production of antibiotic substances by natural variants of the marine bacterium Vibrio Fischeri]. 273 Feb 20

An improved rat anterior pituitary primary cell culture technique for studying GH-releasing activity of human pancreatic GH-releasing factor (hpGRF) and its analogs is described. Male pituitaries, dispersed by a combination of trypsin digestion and mechanical agitation, were plated at a density of 200,000 cells per well and cultured for 4 days. The attached cells were then stimulated with synthetic hpGRF which was comprised of the first 29 residues of the larger, originally isolated forms and which was amidated at the C-terminal (hpGRF-29). Analogs of hpGRF-29 which were modified in positions 1, 2, 3, or 7, and other secretagogues were similarly tested. Medium was collected after 3 h, and secreted hormone was measured by RIA. The cells were extremely sensitive to hpGRF-29 stimulation, and this effect was specific. The minimal effective dose of hpGRF-29 was an unprecedented 0.4 X 10(-15)M. No stimulation of LH, FSH, or PRL by hpGRF-29 was observed. Bombesin and vasoactive intestinal peptide were ineffective in stimulating GH release. [D-Trp6]LHRH (a potent LHRH agonist), also did not release GH but did stimulate secretion of LH and FSH at doses ranging from 0.4 X 10(-10)M to 1.0 X 10(-9)M. Responses of the cells to hpGRF-29 analogs were characterized by distinct heterologous dose-response curves. [D-Ala2]hpGRF-29 was 50 times more active than its parent 29-amino-acid peptide. [D-Thr7]hpGRF-29, another analog that differed from hpGRF-29 by the insertion of a D-isomer for the naturally occurring L-residue, was about 10,000 times less effective in stimulating GH secretion than was hpGRF-29 itself. Potencies of these and other analogs with respect to GH release in vitro were similar to those estimated in vivo. Thus, this primary cell culture provides an extremely sensitive, selective, and reproducible system for studying hpGRF structure-activity relationships. Further, such tremendous sensitivity to hpGRF can provide a system to study changes in pituitary sensitivity to hpGRF during different physiological states.
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PMID:An extremely sensitive in vitro model for elucidating structure-activity relationships of growth hormone-releasing factor analogs. 285 74

Demembranated ciliated cell models are useful for studying mechanisms responsible for the regulation of ciliary coordination and waveform. This paper describes procedures for isolating ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, their subsequent demembranation by Triton X-100, and their reactivation with MgATP to produce highly motile, coordinated, ciliated cell models. Reactivation of cell models with a high degree of mechanochemical coupling depended on avoiding mechanical damage and maintaining optimal conditions during all stages of isolation and reactivation. Highly motile models were prepared from cells incubated in trypsin, treated briefly with EDTA, separated by gentle agitation, and concentrated by centrifugation at low gravitational forces. Optimal demembranation and reactivation conditions were similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models were reactivated when provided with MgATP and 90-95% beat with coordinated waves. The ciliary tufts beat at frequencies within the range measured in living cells and their reactivated motility was stable for at least 30 min at constant MgATP. These highly coupled models were used to show (1) that development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 microM) than that required to initiate motility per se (2-10 microM; (2) that outer dynein arms may not contribute to beat frequency at substrate concentrations below 35 microM; and (3) that vanadate has effects both on beat frequency and coordination of the tufts.
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PMID:Isolation of newt lung ciliated cell models: characterization of motility and coordination thresholds. 293 51

The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.
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PMID:Flow cytometric evaluation of cell dispersion from human head and neck tumors. 299 Aug 34

It was recently reported (Endoh et al. 1981, Exp Cell Biol 49:272-277) that conditioned medium of neonatal mouse brain (CM-NB) inhibited the growth of mouse neuroblastoma cells. In this work we fractionated CM-NB by size exclusion high performance liquid chromatography, and separated two active principles (28,000 and 62,000 daltons) Each or a combination of the 28,000 and 62,000 dalton fractions showed a differential inhibitory effect on DNA synthesis or clonal growth of the three human lung cell lines: the normal diploid fibroblast WI38 cells were less susceptible than their simian virus 40-transformed VA13 cells and carcinoma A549 cells. This preferential growth-inhibition of malignant cells was also observed for rat fibroblast 3Y1 and its simian virus 40-transformed W3Y cells, and for two other normal and five other malignant cell lines. The growth-inhibitory activity of CM-NB or the 28,000 and 62,000 dalton fractions was lost by pronase, trypsin, tetrahydrofuran, acetonitrile, or dithiothreitol in the presence of guanidine, and also labile to heat, vigorous agitation, or freeze-thawing. The activity was also found in the conditioned medium of prenatal mouse brain, but not in either the conditioned medium of the adult brain and of the secondary culture of the neonatal brain, or in the homogenate and rinsing fluid of the neonatal brain. Thus the mouse brain at the terminal stage of ontogenesis liberates proteinaceous factors, which exhibit a preferential growth-inhibition of tumor or transformed cells and act on malignant cells of human and rodent origin.
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PMID:Inhibition of tumor cell growth by protein factors derived from the developing mouse brain. 407 18

Treatment of the neurointermediate lobe of the rat pituitary gland with mechanical agitation in the presence of trypsin and DNAse results in a preparation of cells which secrete alpha MSH and synthesize cAMP. The receptors for catecholamines present in the intact intermediate lobe remain functional on the dispersed cells. Thus, stimulation of a beta-adrenoceptor with (l)isoproterenol enhances the secretion of alpha MSH. This receptor is stereospecific and responds to low concentrations of isoproterenol (EC50 = 0.4 nM). Activation of a beta-adrenoceptor also increases the accumulation of cAMP. Furthermore, dopamine inhibits the basal release of alpha MSH but has no effect on basal levels of cAMP. In addition, dopamine inhibits the isoproterenol-enhanced release of alpha MSH as well as the isoproterenol-induced accumulation of cAMP.
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PMID:Release of alpha-melanocyte-stimulating hormone from dispersed cells of the intermediate lobe of the rat pituitary gland: involvement of catecholamines and adenosine 3',5'-monophosphate. 624 51

Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].
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PMID:Porcine liver succinyltrialanine p-nitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme. 636 Oct 13

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginal discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginal discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginal discs, mechanical stirring in Ringer low in Ca2+ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.
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PMID:Removal of insect basal laminae using elastase. 643 33

Mitotic apparatuses (MAs) isolated from sea urchin eggs contained clusters of granular material in their centrospheres. After cold treatment and mild agitation, the MA fraction formed asters when combined with tubulin. Many microtubules grew from isolated centrospheres most of which were covered with astral residues. Homogenization of the isolated MA fraction dispersed the centrospheres which broke into fragments or into aggregates of small granules that formed small asters when tubulin was added. Electron microscopy showed that more than ten microtubules were nucleated from a granular aggregate composed of several approximately 90-nm granules. The aster-forming activity was lost with time when the MAs were kept at 0 degree C. Only glycerol stabilized this activity. The aster-forming activity also was heat labile and trypsin sensitive, but it was resistant to RNase treatment. When the dispersed MAs were extracted with a buffer solution of high ionic strength, aster-forming activity was recovered only in the extract; that is, when the extract had been dialyzed against a solution of low ionic strength, the fine granules self assembled and retained their aster-forming ability.
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PMID:Aster formation in vitro is nucleated by granules isolated from the mitotic apparatus. 650 68


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