A method to detect proteinase activity using unprocessed X-ray films

Cheung, Ambrose L.; Ying, Patrick; Fischetti, Vincent A.

doi:10.1016/0003-2697(91)90037-t

Cited by 30 publications

(23 citation statements)

References 7 publications

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“…Proteolysis tests. Two assays were executed for determination of proteolytic activities, a qualitative assay based on clearance of unprocessed X-ray film material (8) and a quantitative one measuring hydrolysis of dye-coupled collagen (7,17).…”

Section: Methodsmentioning

confidence: 99%

A Regulator of Aspergillus fumigatus Extracellular Proteolytic Activity Is Dispensable for Virulence

Bergmann¹,

Hartmann²,

Cairns

et al. 2009

Infect Immun

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Virulence of the fungal pathogen Aspergillus fumigatus is in part based on the saprophytic lifestyle that this mold has evolved. A crucial function for saprophytism resides in secreted proteases that allow assimilation of proteinaceous substrates. The impact of extracellular proteolytic activities on the pathogenesis of aspergillosis, however, remains controversial. In order to address this issue, characterization of a conserved regulatory factor, PrtT, that acts on expression of secreted proteases was pursued. Expression of PrtT appears to be regulated posttranscriptionally, and the existence of an mRNA leader sequence implies translational control via eIF2␣ kinase signaling. Phenotypic classification of a prtT⌬ deletion mutant revealed that expression of several major extracellular proteases is PrtT dependent, resulting in the inability to utilize protein as a nutritional source. Certain genes encoding secreted proteases are not regulated by PrtT. Most strikingly, the deletant strain is not attenuated in virulence when tested in a leukopenic mouse model, which makes a strong case for reconsidering any impact of secreted proteases in pulmonary aspergillosis.

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Section: Methodsmentioning

confidence: 99%

A Regulator of Aspergillus fumigatus Extracellular Proteolytic Activity Is Dispensable for Virulence

Bergmann¹,

Hartmann²,

Cairns

et al. 2009

Infect Immun

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“…The gelatin decomposing activity of the extracts was examined with the use of overexposed and developed photographic films (Henry et al 1974;Cheung et al 1991). A Kodak film was placed in a slide frame and 3 µl of a sample mixed with 3 µl of the appropriate buffer solution was put onto each piece of film.…”

Section: Methodsmentioning

confidence: 99%

Activity of selected hydrolytic enzymes from leeches (Clitellata: Hirudinida) with different feeding strategies

2009

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Hydrolase activities of extracts from different parts of the bodies of parasitic (Theromyzon tessulatum, Piscicola geometra) and predatory (Erpobdella octoculata, Glossiphonia complanata) leeches were examined. The highest activity was detected in the extracts from sections containing the intestine. Hydrolase activities in the crop and intestine of parasitic leeches were higher than in predatory leeches. The high activity of most of hydrolases in those segments may indicate the intensity of digestion and absorption processes in leeches. A lack of trypsin activity and low chymotrypsin activity are likely to result from the presence of inhibitors of these enzymes. The high activity of the majority of the analyzed hydrolases in extracts derived from the head segment of predatory leeches enables, through digestion of tissues, their easy access to physiological fluids of a host. In turn, in extracts from the head segment of predatory leeches, only four hydrolases were shown to be active. Lipase activity was not found in any of the samples, while α-galactosidase activity was found only in extracts from the head segment of T. tessulatum and P. geometra. Trypsin activity was detected in the extract from the intestine contents of H. sanguisuga and in the extract from the head segment of P. geometra. The results demonstrate the presence of majority of hydrolases occurring in other animals in the alimentary tract of leeches. The study also shows that the crop of leeches is not only a food reservoir, but also the site where digestion and absorption processes take place.

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“…To eliminate the prospect that the enzymatic degradation of the 5D4 epitope by keratanase II results from nonspecific protease activity in the keratanase II preparation, we used the gelatin coating of unprocessed X-ray film as a protease substrate (Cheung et al, 1991). Although purified trypsin clearly digests the gelatin coating at both concentrations tested, the protease inhibitor mixture used throughout our studies blocked this degradative activity (Fig.…”

Section: Sds-page Analysis Of 5d4 Immunolabeling In Developing Brainmentioning

confidence: 98%

“…Keratanase II was tested in the presence and absence of protease inhibitors at the same activity (1 mU/µg protein) as that used in the SDS-PAGE studies. Trypsin, which readily digests the film gelatin, was used as a positive control (Cheung et al, 1991). We prepared six test solutions in 0.1 M sodium acetate buffer (pH 6.3): trypsin (2 µg/ml and 10 µg/ml), trypsin (10 µg/ml) in buffer containing the protease inhibitor mixture, buffer alone, keratanase II, and keratanase II in buffer containing the protease inhibitor mixture.…”

Section: Test For Nonspecific Protease Activitymentioning

confidence: 99%

“…We tested for nonspecific protease activity in the keratanase II preparation with the method of Cheung et al (1991), which uses the gelatin coating of unprocessed X-ray film as a substrate. Keratanase II was tested in the presence and absence of protease inhibitors at the same activity (1 mU/µg protein) as that used in the SDS-PAGE studies.…”

Section: Test For Nonspecific Protease Activitymentioning

confidence: 99%

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Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains

1997

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Proteoglycans influence axonal outgrowth in several experimental paradigms, and their distribution during development suggests a role in axon guidance. We have used a monoclonal antibody, 5D4, that recognizes an epitope on sulfated keratans (KS), to define the distribution of keratan sulfate proteoglycans (KSPGs) in the developing thalamus and cortex of the rat. During development, 5D4 immunolabeling is present on thalamic axons as they grow through the internal capsule and subplate but is not present in the adjacent pathway for cortical efferent axons. Individual thalamic nuclei differ markedly in their expression of KSPGs; these distinctions persist throughout the period of developmentally regulated expression. Major cortical domains also differ in their expression of KSPGs, which are expressed throughout medial (cingulate and retrosplenial) cortex well before neocortex. Immunolabeling for KSPGs diminishes 2 weeks after birth; in the adult it is associated with small glia. The 5D4 epitope is present on several KSPGs (320, 220, and 160 kD) on Western blots during development but only in a broad 200-kD band in adult brain. Immunolabeling is degraded on sections and Western blots by keratanase II but not by keratanase I or chondroitinase ABC, confirming that the antibody recognizes KS. Bands identified by 5D4 on Western blots differ from those identified by antibodies to known KSPGs (aggrecan, claustrin, SV2, ABAKAN, phosphacan-KS), indicating that 5D4 is labeling KSPGs not previously described in the brain. The selective expression of KSPGs during development suggests that they may be a part of the molecular identity of thalamic nuclei and cortical domains that defines their connectivity.

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A method to detect proteinase activity using unprocessed X-ray films

Cited by 30 publications

References 7 publications

A Regulator of Aspergillus fumigatus Extracellular Proteolytic Activity Is Dispensable for Virulence

A Regulator of Aspergillus fumigatus Extracellular Proteolytic Activity Is Dispensable for Virulence

Activity of selected hydrolytic enzymes from leeches (Clitellata: Hirudinida) with different feeding strategies

Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains

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