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)

Prokallikrein was activated by trypsin and by alpha-chymotrypsin, but not by proteases, such as plasmin, thrombin, urokinase, carboxypeptidase B, papain, elastase, pepsin, and cathepsin D. Moreover, rat fresh serum did not activate prokallikrein. Maximum activation of prokallikrein by trypsin was obtained at the concentration of 10 micrograms to 1 mg per ml in PBS and that by alpha-chymotrypsin was at the concentration of 5 mg per ml. The enzymic properties of trypsin-activated and alpha-chymotrypsin-activated kallikreins were identical with those of active kallikrein in the kidney.
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PMID:Activation of prokallikrein in the rat kidney by proteases. 637 43

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.
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PMID:In vitro development of the embryonic mouse inner ear following exposure to trypsin. 661 20

A new continuous cell strain (CaPi) was established from normal pituitary tissue of carp, Cyprinus carpio L. Cells of a fractionated dispersion (0.25% trypsin-PBS, 10 degrees C) of pituitaries formed monolayer (fibroblast- and epithelial-like-cells) at 25 degrees C using Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum and non essential amino acids. The continuous cell strain, designated CaPi, was obtained by an enzymatical selection of epitheloid cells from the monolayer of the primary pituitary cell culture. This culture has been subcultured 70 times over a period of 24 months. CaPi-cells multiply over a temperature range of 15-35 degrees C with an optimum growth temperature of 30 degrees C at a seeding density of 1.7 X 10(6) cells/ml. Chromosome analysis indicates the cells are heteroploid and show modal numbers of 47 chromosomes. The CaPi cell line is susceptible to fishviruses (VHSV, PFR, SVCV) producing cytopathic effects (CPE). However, IPNV replicates without visible cell alterations.
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PMID:[Establishment of a fish-virus susceptible cell strain from the pituitary gland of carp (Cyprinus carpio L.)]. 667 52

1. We describe a simple and accurate colorimetric adhesion assay (CAA) and illustrate the assay by measuring the adhesion of mouse thymocytes to mouse 2BH4 cells. 2. The assay is based on the crystal violet staining of thymocytes adhered to a subconfluent layer of 2BH4 cells (plated at 2 x 10(4) cells/well for 24 h). The optimal incubation time was shown to be 1 h and washing in PBS of non-bound and non-specifically bound thymocytes is the critical step for the precision and accuracy of the assay. 3. Saturation curves were obtained for thymocytes adhered to plated 2BH4 cells. The blank (only 2BH4 cells) was near 0.200 +/- 0.010 (mean +/- SD) and was quite reproducible. As expected, the extent of adhesion was also dependent on the number of plated 2BH4 cells. Standard curves need to be run with each assay for quantitative measurements. The intra-assay and interassay coefficients of variation were 5% and 20%, respectively. 4. The specificity of the reaction was demonstrated by the reduction of adherence by trypsin pretreatment of thymocytes, and the dose-dependent inhibition of adherence by rabbit anti-mouse thymocyte antisera but not rabbit anti-mouse immunoglobulin antisera. 5. The proposed method is simple and requires less effort than the counting of adhered cells with the light microscope and does not require the use of radioactive material as when labelling with Na2(51)CrO4 is utilized.
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PMID:Colorimetric assay for the measurement of thymocyte/thymic stromal cell adhesion. 753 44

The use of chain-specific monoclonal antibodies (MAbs) against keratins in pathology is hampered by their limited staining on formalin-fixed, paraffin-embedded tissue. In the present study, various treatments before immunohistochemistry on paraffin sections were compared, including proteolytic enzymes and microwave antigen retrieval in various solutions. Sections of normal cervical and skin tissue were stained in a three-step immunoperoxidase method, employing a broad panel of MAbs against chain-specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pankeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antigen Retrieval Solution (ARS), a simple detergent solution (DET), PBS, and distilled water (MiQ) were compared. Microwave heating in PBS or MiQ strongly improved staining results. Moreover, microwave pre-treatment in TUF or DET gave excellent and specific staining with the majority of MAbs tested, comparable with or even better than staining obtained on frozen sections. Using microwave antigen retrieval, tissue morphology remained optimal, and only in a very limited number of MAbs did immunoreactivity on paraffin sections fail to be restored. Proteolytic pre-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pre-treatment was able to restore the loss of immunoreactivity, failed to give appropriate staining with proteolytic pre-treatment. Our results show that microwave heating in either TUF or a simple detergent solution before immunohistochemistry is a reliable method for antigen retrieval of chain-specific keratins in formalin-fixed, paraffin-embedded tissues.
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PMID:Immunostaining of chain-specific keratins on formalin-fixed, paraffin-embedded tissues: a comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments. 753 85

Time-resolved anisotropy was utilized to detect nanosecond segmental motions of the band 3 intramembrane domain. Band 3 at lysine 430 was fluorescently labeled in ghost membranes by fluorescein or eosin maleimide treatment of intact human erythrocytes followed by hypotonic lysis. Single lifetimes for fluorescein (3.8-4.1 ns) and eosin (3.2-3.4 ns) were observed. Phase-modulation measurement of anisotropy decay indicated a segmental motion model, r(t) = exp(-t/tau 1c)[r infinity + (ro-r infinity) exp(-t/tau 2c)], defined by rotational correlation times corresponding to band 3 segmental motion (tau 1c, 30-70 ns) and rapid fluorescein motion in its binding pocket (tau 2c, 200-400 ps), and a residual anisotropy (r infinity, 0.23-0.28) describing hindered fluorescein motion. In PBS at pH 7.4, tau 1c, tau 2c, and r infinity were 44 ns, 307 ps, and 0.24, respectively, predicting a steady-state anisotropy of 0.24, in agreement with the measured value of 0.23. Factors that might influence band 3 structure/dynamics were examined. Whereas pH (range 5-10) had little effect on r(t), [NaCl] addition (0-150 mM) remarkably decreased tau 1c from 68 to 44 ns. The decrease in tau 1c correlated with solution ionic strength, and did not depend on osmolality (studied by mannitol addition), or specific anion interactions (comparing Cl, Br, F, SO4, citrate). The ionic strength effect was not observed in fluorescein-labeled carbonic anhydrase and trypsin-cleaved band 3, suggesting a specific effect on intact band 3. Anisotropy decay was relatively insensitive to external lectin or internal 2,3-DPG binding, but was sensitive to temperature, membrane fluidity, urea denaturation, fluid-phase viscosity, and aldehyde fixation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Anisotropy decay measurement of segmental dynamics of the anion binding domain in erythrocyte band 3. 754 17

The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.
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PMID:Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium. 768 17

We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10(-8) M), a total of 4 to 5 x 10(7) cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone resorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID50) was 1.08 x 10(-11) M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable for forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclastic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.
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PMID:Establishment of a rapid bone resorption in vitro assay using previously frozen mouse unfractionated bone cells pretreated with parathyroid hormone. 815 5

Mycoplasma salivarium cells were demonstrated to bind human IgG Fc fragments. The binding capacity was 78.8% enhanced by incubation of the cell suspension in PBS with 0.25% trypsin at 37 degrees C for 1 h, but tended to fall after incubation with higher concentrations of the enzyme, and was 95.5% lower after incubation of the suspension without trypsin. Fc fragments of IgG from rat, swine, sheep, rabbit, goat, cow and mouse also bound to the organism cells with increasing affinity in this order. The affinity of human IgG Fc fragments was almost comparable with those of sheep and rabbit. Antigen specific IgG from goat (specific for gamma-chain of human IgG, and mu-chain of human IgM) and rabbit (specific for whole molecules of goat IgG) bound to the cells. Binding of goat IgG Fc fragment was inhibited in the presence of antigen specific goat IgG (specific for gamma-chain of human IgG). These results suggest that M. salivarium cells bind IgG from a variety of animal species via the Fc fragment.
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PMID:Binding of Fc fragments of IgG from human and seven animal species to Mycoplasma salivarium cells. 829 54

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.
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PMID:Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis. 881 94


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