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)

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.
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PMID:Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae. 846 Dec 93

Groups of sucking Swiss albino mice were inoculated by the intracerebral (i.c.), intraperitoneal (i.p.) or oral route with a trypsin-sensitive avian reovirus (TR1) or a trypsin-resistant (R2) reovirus. The viruses caused a number of effects, the most severe occurring after i.c. inoculation and the least after oral inoculation. They included incoordination and tremors, oiliness of the hair, and retarded growth. Patterns of viral persistence in tissues were similar for the two viruses, with high titres in the brain on days 3 and 6 after i.c. or i.p. injection. Both viruses were still present in the brain 21 days after i.c. injection. No virus was found in any tissue when TR1 was given orally. All groups "seroconverted" except the one infected orally with TR1, but neutralization titres were low. The effects resembled those described for mammalian reoviruses in mice. The results indicate that, for short periods, wild mice may be capable of transmitting avian reoviruses between poultry flocks. Furthermore, in the production of monoclonal antibodies to avian reoviruses in mice, it is possible that pathological changes will occur.
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PMID:Experimental inoculation of mice with trypsin-resistant and trypsin-sensitive avian reoviruses. 944 85

Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditis). The majority of TPO autoantibodies in individual patients' sera interact with a restricted immunodominant region on TPO. Although this region can be mapped, previous studies have failed to localize its position on the TPO molecule. We, therefore, used a footprinting approach that can localize a highly conformational, discontinuous epitope on a very large molecule. Extensive biotinylation ( approximately 15 biotins/molecule protein) of lysine residues on the surface of purified, native TPO resulted in loss of multiple tryptic cleavage sites, as determined by analysis of tryptic polypeptide fragments on reverse-phase HPLC. TPO was then complexed with a monoclonal human autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR1.9, TPO was recovered by gel filtration. A trypsin site, previously observed to be lost after TPO biotinylation, was restored when biotinylation was performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was present in a 30-aa TPO fragment that, by N-terminal sequencing, was found to be K713. Altered recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule involving this region supported the epitope protection data. In conclusion, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. This focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region.
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PMID:Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope. 1114 17

Two groups of commercial Light Sussex hens with no cultural evidence of reovirus infection and very low titres of neutralising antibodies were mated with cockerels from 17 weeks of age. At 27 weeks of age the birds were separated into three groups, and were inoculated intranasally and intravenously with avian reovirus strain R2 which is resistant to trypsin, with strain TR1 which is sensitive to the enzyme or sham-inoculated. Of the eggs laid by the hens infected with strain R2, 13/29 infertile eggs and embryos which fails to hatch were positive for virus, as were 6/70 hatched chicks. Despite this, virus was never isolated from cloacal swabs from the hens. Virus-infected eggs were laid between days 5 to 17 post inoculation (p.i.). Virus was isolated from the liver of all six R2 virus-positive chicks, from the hock joint of four and from the intestine of three. In contrast, for the group infected with the trypsin-sensitive virus TR1, of 120 eggs laid in the 5-week period, virus was isolated once only, from a chick hatched from an egg laid 7 days p.i. This infected chick was one of 83 which hatched and virus was found only in the joint. In a further experiment, two groups of mature SPF hens were inoculated with the reoviruses as above. Cloacal swabs and tissue examination showed greater virus excretion and tissue distribution of R2 than TR1. These results helped to explain the much higher egg transmission rate of R2 than TR1. However, the rate of vertical transmission of chicken reoviruses in nature, where the infectious dose would normally be lower than given here, is likely to be low.
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PMID:Egg transmission of avian reoviruses in chickens: comparison of a trypsin-sensitive and a trypsin-resistant strain. 1864 73

Experiments are described which show how the sensitivity to trypsin of avian reovirus strain TR1 restricts its replication in the intestine of the chicken in comparison with a trypsin-resistant strain R2. Following oral infection with a high dose (5.3 log10 TCID50), the trypsin-sensitive virus generally showed lower titres than the resistant one in all tissues examined. Infection of chicks with strain TR1 via the respiratory route enabled the virus to spread throughout the body and localize in the hock joint, an important target site for reoviruses. Trypsin-sensitive reoviruses might be transmitted via the respiratory route, even though TR1 caused little damage to the respiratory epithelium. Dose-response studies showed that TR1 injected via the footpad can localize in the hock joint after very low doses, but high oral doses (4-5 log10) are necessary for such localization. Intranasal infection was intermediate in effect.
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PMID:Early pathogenesis in chicks of infection with a trypsin-sensitive avian reovirus. 1867 Nov 34

A reovirus (strain TR1) was isolated from the hock joint of a turkey with arthritis. It was rarely excreted in the faeces of poults after oral or intranasal inoculation and only occasionally following footpad inoculation. The virus did not localise in the hock joints or cause lesions. Cyclophosphamide-induced immunosuppression of poults did not modify the infection. Oral inoculation of chicks with a high dose of strain TR1 resulted in joint localisation, of virus and erosive arthritis. Footpad inoculation caused deaths in 13 of 15 chicks by 8 days pi due to hepatic necrosis. Intranasal inoculation of chicks resulted in a delayed and limited faecal excretion. However, joint lesions were most marked in chicks infected via this route. The infectivity of strain TR1 in chick embryo liver cell monolayers was reduced by almost 3.0 log(10) after treatment with 0.01% trypsin for 30 min, but the infectivity of strain R2 was unaffected. Bile salts had no effect on either virus. This trypsin-sensitive reovirus was much more pathogenic for chicks than for poults. The respiratory route may be important in the dissemination of trypsin-sensitive avian reoviruses such as this.
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PMID:A trypsin-sensitive avian reovirus: isolation and experimental infection of poults and chicks. 1867 94