Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assays of serum benzylamine oxidase (BzAO) have led some workers to postulate a relationship between elevated BzAO activity and diseases characterized by proliferating connective tissue. The present study was designed to determine whether BzAO activity of a cellular tissue is also affected. BzAO was assayed in homogenates of normal and atherosclerotic human aortae. Characterization done in normal aortae showed that BzAO is not a classical monoamine, diamine, polyamine, or lysyl oxidase, nor is it a ceruloplasmin. The enzyme is heat stable at 60 degrees C and is associated primarily with the microsomal fraction on density centrifugation. Compared with phenylethylamines and indoleamines, benzylamine is the best substrate. BzAO is sensitive to inhibition by hydrazines and chymotrypsin but not trypsin, and is insensitive to Triton X-100 and sulfhydryl-group blockade. BzAO activity of atherosclerotic plaque (expressed per gram wet weight or per milligram protein) was decreased markedly compared to that in adjacent, nonplaque regions and in normal aortae. However, on a per milligram DNA basis, the BzAO activity of plaque did not differ from that of nonplaque tissue. We conclude that there is a decreased cell population density in plaque, a contention supported by kinetic analysis. Plaque BzAO showed a decreased Vmax with no change in the Km of benzylamine compared with nonplaque tissue. Thus, if a relationship exists between BzAO activity and proliferating connective tissue, it is not apparent at the level of the cellular enzyme in atherosclerotic aortae of man.
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PMID:Benzylamine oxidase in normal and atherosclerotic human aortae. 683 47

Plaque formation, replication, and related cytopathic functions of the enteropathogenic bovine coronavirus strain L9 in bovine fetal thyroid (BFTy) and bovine fetal brain (BFB) cells were investigated in the presence and absence of trypsin. Plaque formation was enhanced in both cell types. Plaques reached a size with an average diameter of 5 mm within 4 days with trypsin in the overlay, whereas their diameter remained less than 1 mm at this time after plating without trypsin in the overlay. Fusion of both cell types was observed 12 to 18 h after infection when trypsin was present in the medium. Fusion was not observed in infected BFB cell cultures and was rarely observed 48 h after infection of BFTy cells maintained with the trypsin-free medium. The largest polycaryons formed had 15 to 22 nuclei. They then lysed and detached. Cell fusion depended on de novo synthesis of hemagglutinin and infectivity. Fusion from without was not observed. Virus produced under trypsin-enhancing conditions accompanied by cell fusion did not lyse mouse erythrocytes that reacted with L9 coronavirus hemagglutinin. Trypsin-treated, infected BFTy cultures produced coronaviral particles that excluded stain from the envelope confinement. These virions had uniformly shorter surface projections than did the viral forms generated by trypsin-free cell cultures.
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PMID:Enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment. 722 3

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaque-reduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.
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PMID:Development and optimization of plaque assays for rat coronaviruses. 768 80

Various factors influencing plaque formation of Chuzan virus in BHK-21 cell monolayers were studied and a practical method for plaque assay was developed. On addition of trypsin (5 micrograms/ml) and/or diethylaminoethyl (DEAE)-dextran (50 micrograms/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 micrograms/ ml DEAE-dextran was added to the diluent and overlay medium, plaques were produced in about 10-fold higher numbers than without trypsin and DEAE-dextran. Based on these results, a practical plaque assay method for Chuzan virus was established. Using this method, one-step growth of Chuzan virus was performed at an input multiplicity of 25 plaque-forming units (PFU) per cell. Cytopathic effects were first observed at 7.5 h post-inoculation (p.i.), and were complete at 12 h p.i. The titre of cell-associated virus, after gradual decline during the first 3 h of incubation, showed a rise within 4.5 h p.i. and a rise to a plateau of 10(6.3)PFU/0.2 ml at 12 h p.i. By indirect immunofluorescence, virus-specific antigen was detected in the cytoplasm of the cells at 4.5 h p.i., and all the cells fluoresced at 6 h p.i. Haemagglutination activity was first detected in infected whole cultures at 7.5 h p.i. reaching a plateau of 1:64 at 15 h p.i. Plaque formation and haemagglutination by the virus were specifically inhibited by antisera against the original and the plaque-cloned virus.
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PMID:Sensitive plaque assay and propagation of Chuzan (Kasba) virus, a Palyam serogroup orbivirus, in BHK-21 cells. 879 95

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.
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PMID:Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. 980 58

Smoking has been identified as a risk factor for development of periodontal disease and a strong indicator for treatment failure in periodontal patients. This study examined 172 patients categorized as current smokers (n=55), previous smokers (n=38) or individuals that had never smoked (n=79). A total of 670 interproximal plaques collected with a wooden toothpick were analyzed for hydrolysis of the synthetic trypsin substrate benzoyl-DL-arginine naphthylamide (BANA). About 95% of the BANA hydrolysis by plaque is due to the presence of one or more of the periodontopathogens, P. gingivalis, T. denticola or B. forsythus. Gingival health was measured using the papillary bleeding score (PBS). Current smokers had less gingival bleeding than previous smokers or those who had never smoked (20% versus 41% and 25%, respectively). Plaque removed from non-bleeding sites in current smokers were 11x more likely to have a positive BANA reaction when compared to plaque removed from non-bleeding sites in individuals who never smoked. A significant positive relationship exists between smoking and colonization by the BANA periodontopathogens. Smoking may select for these periodontopathic species in the plaque and may be one reason why smoking is a risk factor in periodontal disease development.
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PMID:The prevalence of BANA-hydrolyzing periodontopathic bacteria in smokers. 1059 10

The myxoma virus M131R gene encodes a catalytically inactive homolog of cellular Cu-Zn superoxide dismutase (SOD1) and this 17,786 Da protein is a major virion component. We have used matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS) to study the effect(s) of deleting the gene on virion composition and structure. This approach confirmed that the M131R gene product is an abundant virion component. This conclusion was based upon the ready detection of a 1805.3 Da peptide released from the N-terminus of the myxoma SOD1 protein by mild trypsin treatment, as well as the detection of a 17,790 Da protein in HPLC fractionated virus extracts, which subsequently yielded M131R-encoded tryptic peptides. Neither peptide nor protein was detected in particles bearing a genome encoding an M131RDelta deletion mutation. Curiously, more proteins and tryptic peptides were detected when M131RDelta mutant virions were subjected to MALDI-TOF MS analysis compared with wild-type virus particles. This suggested that particles assembled in the absence of myxoma SOD protein are structurally unstable. Plaque analysis confirmed this conjecture by showing that SOD-deficient MYX particles are unusually heat labile and trypsin sensitive. Mutant Shope fibroma virus exhibited the same phenotype. Thus a previously unappreciated feature of MALDI-TOF MS is that the method can sometimes detect alterations in virion stability.
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PMID:MALDI-TOF mass spectroscopy detects the capsid structural instabilities created by deleting the myxoma virus cupro-zinc SOD1 homolog M131R. 1548 22

With the emergence of new viruses, such as the SARS virus and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. Sendai virus causes a localized respiratory tract infection in rodents, while a mutant, F1-R, causes a systemic infection. It has been suggested that two determinants are responsible for the systemic infection caused by F1-R [Okada et al (1998) Arch Virol 143:2343-2352]. The primary determinant of the pantropism is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R, which allows the virus to undergo multiple rounds of replication in many different organs, whereas wild-type virus can only undergo multiple rounds of replication in the lungs. The enhanced cleavability of F1-R F was previously attributed to an amino acid change at F115 that is adjacent to the cleavage site at amino acid 116. Secondly, wild-type virus buds only from the apical domain of bronchial epithelium, releasing virus into the lumen of the respiratory tract, whereas F1-R buds from both apical and basolateral domains. Thus, virus is released into the basement membrane where it can easily gain access to the bloodstream for dissemination. The microtubule disruption is attributed to two amino acid differences in M protein. To confirm that the F and M gene mutations described above are solely responsible for the phenotypic differences seen in wild-type versus F1-R infections, reverse genetics was used to construct recombinant Sendai viruses with various combinations of the mutations found in the M and F genes of F1-R. Plaque assays were performed with or without trypsin addition. A recombinant virus containing all F1-R M and F mutations formed plaques in LLC-MK2 cells and underwent multiple cycles of replication without trypsin addition. To clarify which mutation(s) are necessary for plaque formation, plaque assays were done using other recombinant viruses. A virus with only the F115 change, which was previously thought to be the only change important for plaque formation of F 1-R F, did not confer upon the virus the ability to form plaques without the addition of trypsin. Another virus with the F115 and both M changes gave the same result. Therefore, more than one mutation in the F gene contributes to the ability of F1-R to form plaques without trypsin addition.
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PMID:Mutations in Sendai virus variant F1-R that correlate with plaque formation in the absence of trypsin. 1583 52

In this study, a 15-mer phage display peptide library was employed to pan against human rotavirus immobilized on solid phase. 4 different peptides were selected and could bind with rotavirus particles specifically. Plaque reduction neutralization test and MTT analysis results indicated that 3 of the peptides can inhibit rotavirus infecting in vitro. A peptide which sequence is QSNPIHIITNTRNHP showed the best efficiency--93% neutralization infectivity. Two other peptides, A and B, showed 40% and 50% neutralization infectivity respectively. Amino sequence analysis results indicate the 3 peptides containing 2 conserved motifs: SNPIHII and NIP. No putative trypsin hydrolysis site was found in C peptide, however, 4 and 3 potential sites were found in A and B peptides respectively. Using trypsin inhibitor, both A and B peptides showed the similar antiviral effect as that of C peptide. It suggests that the intactness of the 2 conserved motifs play an important role in counteracting virus infection. According to the results of this study, peptide C is hopeful to be exploited as an antiviral peptide drug.
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PMID:[Screening for peptides of anti-rotavirus by phage-displayed technique]. 1757 83

Metagenomic techniques are used to analyse bacterial communities allowing both culturable and unculturable species to be represented. However, the screening of oral metagenomic samples can be hindered by high animal host DNA content. This study evaluated methods for the reduction of human DNA concentrations within oral metagenomic samples. Plaque samples were collected from 27 patients presenting with periodontal disease and treated to remove human DNA using either selective lysis of eukaryotic cells at several buffer concentrations or differential centrifugation after treatment with trypsin and/or detergents. Human and bacterial DNA levels were determined by quantitative polymerase chain reaction (qPCR). The human DNA content of plaque extracts was significantly reduced by all treatments compared with an untreated control (P < 0.05). However, differential centrifugation simultaneously reduced the bacterial DNA content unless samples were pretreated with a detergent. Observations of Gram stained samples that were processed using differential centrifugation without detergent suggest that many bacteria remain adhered to human cells. An approach that uses differential centrifugation in parallel with selective lysis is recommended to fully represent the oral microbiota in metagenomic samples, including those tightly adhered to human cells and more delicate bacteria such as Mycoplasma.
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PMID:Selective removal of human DNA from metagenomic DNA samples extracted from dental plaque. 2129 92


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