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)

There are several methods available for the production of Schwann cell cultures from fetal and neonatal peripheral nervous tissue. We have investigated methods for producing Schwann cell-rich cultures from adult tissue. Dorsal root ganglia from normal adult cats were used to initiate explant cultures or subjected to primary dissociation. The resulting cultures were compared in terms of growth, the proportions of fibroblastic and Schwann-like cells in primary cultures and the effects of subculture on the relative frequency of these cell types. We found that excision and transfer of explanted ganglion pieces after 14 days in culture produced a secondary outgrowth rich in small, bipolar, spindle-shaped Schwann-like cells. Subculture of this outgrowth produced secondary cultures of predominantly Schwann-like cells with typical spindle-shaped morphology. The use of antimitotic agents in the media to inhibit fibroblast growth was not observed to be necessary or beneficial with this adult tissue. Primary dissociation of ganglia with enzymes (trypsin or collagenase) and mechanical agitation was even more effective in producing secondary cultures and cell lines that were, by morphological criteria, predominantly or exclusively Schwann-like cells. One of these Schwann-like cell lines, designated GSA, has been carried over 24 subcultures while retaining characteristic Schwann cell morphology. Cells of this line have been examined by scanning and transmission electron microscopy. Karyotype analysis indicates a chromosome complement consistent with the species of origin, a normal cat.
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PMID:Non-neuronal cell cultures from dorsal root ganglia of the adult cat: production of Schwann-like cell lines. 730 4

Sperm flagella of the sea urchin Tripneustes gratilla beat with asymmetrical bending waves after demembranation with Triton X-100 in the presence of EGTA and reactivation at pH 8.1 with 1 mM ATP in the presence of 2 mM MgSO4. Addition of 0.1--0.2 mM free Ca2+ to these reactivated sperm induces 70--95% of them to become quiescent. This quiescence can be reversed by reduction of the free Ca2% concentration with EGTA, or by dilution to reduce the MgATP2- concentration below 0.3 mM. The quiescent waveform is characterized by a sharp principal bend of approximately 5.6 rad in the proximal region of the flagellum, a slight reverse bend in the midregion that averages approximately 0.3 rad, and a principal bend of approximately 1.1 rad in the tip. The quiescent sperm are highly fragile mechanically, and disruption, including microtubule sliding, occurs spontaneously at a slow rate upon standing or immediately upon gentle agitation. Mild digestion by trypsin causes a gradual appearance of normal, symmetrical flagellar beating. Addition of increasing concentrations of vanadate to quiescent sperm causes a graded decrease in the proximal bend angle, with 50 micrometers vanadate reducing it to approximately 2.6 rad. In the presence of 0.1 mM free Ca2% and 10 micrometers vanadate, a characteristic, crescented stationary bend is induced in the demembranated sperm, without intermediate oscillatory beating, by the addition of either 0.1 or 1 mM ATP. In the absence of vanadate, these two concentrations of ATP produce asymmetric beating and quiescence, respectively. The results support the hypothesis that quiescence in live sperm is induced by an elevated concentration of intracellular Ca2%. In addition, they demonstrate that bending can occur in flagella in which oscillatory beating is inhibited and emphasize the close relationship between asymmetric beating and quiescence.
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PMID:Calcium-induced quiescence in reactivated sea urchin sperm. 735 Jan 65

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.
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PMID:Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis. 779 76

Sporulated oocysts of a field strain (FS-111) and a laboratory strain (WIS) of Eimeria tenella were exposed to 0, 50, 100, 150, or 200 Gy of gamma-radiation from a 60Co source. Irradiated oocysts of WIS and FS-111 were not significantly more fragile after irradiation as shown by the release of sporocysts after 5-105 s of vortex agitation with glass beads. Excystation was normal in both strains after treatment of the sporocysts with trypsin and sodium taurodeoxycholate, even in groups exposed to 200 Gy of radiation. Sporozoite release from irradiated sporocysts was more rapid than that from unirradiated sporocysts, primarily because of a shorter lag phase during the first 30 min. Irradiated sporozoites were slower to parasitize cultured chick kidney cells than were control sporozoites (4 h postinoculation), but after 24 h there was no significant difference (P < 0.05) between irradiated and control groups except for the WIS treated with 200 Gy. After 48 h, developing schizonts were reduced by 77-94% on exposure to 50-200 Gy. Strain FS-111 did not develop as well as WIS in vitro, but the effect of irradiation was similar. When irradiated oocysts of WIS or FS-111 were inoculated into chickens the prepatent period was unaffected, but fewer oocysts were produced, lesion scores were lower, and the weight gain was less strongly affected in proportion to the doses of radiation. These results suggest that the effects of radiation damage were largely confined to the mechanism of nuclear and cellular reproduction rather than other physiological processes.
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PMID:Biological effects of gamma-irradiation on laboratory and field isolates of Eimeria tenella (Protozoa; Coccidia). 966 Jan 31

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.
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PMID:Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer. 1039 3

High cell density cultivation has been one of the most effective ways to increase cell as well as the product yields. The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as fusion protein with 6x histidine residues under the transcriptional regulation of the T5 promoter in Escherichia coli. Various strategies were tried to scale up the expression of the recombinant lethal factor by bioprocess optimization using fed batch culture technique in a 14 litre fermentor. The media, a defined mixture of salts, trace elements, vitamins, etc. along with a specified carbon source was used for the growth. The pH of the media was maintained at 6.8 while the temperature was changed from 37 to 28 degrees C during the cultivation. During the growth and induction phases, the DO was maintained above 20% by automatic control of agitation. The specific growth rate was controlled by utilizing an exponential feeding profile determined from mass balance equations. As a result of control of specific growth rate at two different levels, there was about twenty five fold increase in biomass compared to the biomass in the shake flask. E. coli cells yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa. The protein was purified to homogeneity using metal chelate affinity chromatography, followed by anion exchange on FPLC using Mono-Q column. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinity. The recombinant LF resembled the LF purified from B. anthracis in the macrophage lysis assay, using a murine macrophage cell line J774A.1 sensitive to anthrax toxin. It was possible to achieve a yield of 50 mg of the purified protein from 1 litre culture broth.
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PMID:Enhanced expression of the recombinant lethal factor of Bacillus anthracis by Fed-Batch culture. 1146 55

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.
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PMID:Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins. 1257 67

Aphids feed on a protein-poor diet and are insensitive to several serine protease inhibitors. However, among the Bowman-Birk family of plant trypsin inhibitors (BBI), some members display significant toxicity to the pea aphid Acyrthosiphon pisum. A BBI isoform purified from pea seeds (PsTI-2) displays an IC50 of 41 microM and a LC50 of 48 microM at 7 days. Our data show that the chymotrypsin-directed active site from these bifunctional inhibitors is responsible for this activity, and that artificial cyclic peptides bearing the Bowman-Birk anti-chymotrypsin head induce much greater toxicity and growth inhibition than their anti-trypsin counterparts. The toxic syndrome included a rapid behavioural response of aphids on diets containing the toxic peptides, with induced restlessness after only 1 h of exposure to the chymotrypsin inhibitor. Nevertheless, chymotrypsin activity was not detected in aphid guts, using two chromogenic chymotrypsin substrates, and the physiological target of the chymotrypsin inhibitor remains unknown. These data show for the first time that plant chymotrypsin inhibitors, still widely unexplored, may act as paradoxical toxicants to aphids and serve as defensive metabolites for phloem-feeding insects.
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PMID:Toxicity to the pea aphid Acyrthosiphon pisum of anti-chymotrypsin isoforms and fragments of Bowman-Birk protease inhibitors from pea seeds. 1260 15

A series of experiments on various factors which induce optimal in vitro excystation of the metacercariae of Metagonimus yokogawai isolated from the fish, Plecoglossus altivelis was conducted and the following results were obtained. 1)The metacercariae used in this experiment were isolated by the digestion technique therefore all of them were pretreated with the acid-pepsin solution before being applied to the various tests. 2)No excystation occurred when the metacercariae were placed in a salt solutions such as physiological saline, Tyrode solution and Veronal, Tris buffers alone or in combination. 3)The metacercariae underwent complete excystation in the trypsin and pancreatin solution in Tris buffer within an hour at 38 degrees C. The best results were obtained in 0.8-0.9% trypsin solutions, pH 8.0-8.6 and at 38-40 degrees C, approximately one hundred per cent excystation occurred in 40 minutes. Not only temperature but also hydrogen ion concentration played an important role causing excystation of the metacercariae in trypsin-Tris buffer solution. However, bile salts were not responsible for the excystation. 4)Agitation effect on the excystation was tested as a mechanical stimulus and it was found that the shaking stimulus accelerated the excysting mechanism, compared with the metacercariae on which it was not imposed. It is concluded that the metacercariae pretreated in the acid pepsin solution demonstrates an essential requirement for the enzyme solution such as trypsin or pancreatin, provided with the optimum conditions of temperature and hydrogen ion concentration in excysting medium.
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PMID:Study On Metagonimus Yokogawai (Katsurada, 1912) In Korea: II. The In Vitro Excystation Of Metacercariae. 1291 12

In spite of the widespread use of proteins (casein, peptone, etc.) and protein fragments as a substrate for the proteolytic enzymes, a substrate prepared from dyes that adsorb onto appropriate materials, such as wool and cotton, are also used for enzyme activity determination. In the point of view of this thought, it was our aim to develop the substrates which are easily and economically obtainable and also environmentally safer for the frequently used proteolytic enzymes, such as subtilisin carlsberg, trypsin, chymotrypsin, and protease type XVI and, if possible, to prepare the specific substrate at least for one of these enzymes. For this aim, wool was dyed with natural dyes such as juglone, lawsone, berberine, and quercetin. The optimum pH, incubation time, and agitation rate were determinated. The results indicate that, of all the tested enzymes on wool-dye complex as an insoluble substrate, the most appropriate complex was found to be wool-lawsone complex.
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PMID:Dyeing of wool fibres with natural dyes: effect of proteolytic enzymes. 1670 32


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