Histone proteolysis in fish erythrocyte nuclei

The erythrocyte histones of trout, carp, white sucker, and chicken are subject to very different levels of autolytic activity. Carp erythrocyte histones extracted from typical nuclear preparations suffer extensively from degradation; histones 1, 5, and 3 (H1, H5, and H3 respectively) are preferentially cleaved and characteristic peptides designated P1, P2, and P3 appear during the course of proteolysis. Generally, erythrocytes from different fish species yield highly disparate proportions of H1 and H5, but this is not a consequence of the variable levels of proteolytic activity in these species.Phenylmethylsulfonyl fluoride (PMSF) (1.0 mM) was found to be superior to 50 mM sodium bisulfite as a protease inhibitor and was well suited for use in media employed for cell washes and the isolation of nuclei. Nonetheless, in carp erythrocytes residual protease activity (qualitatively the same as the uninhibited activity) persists even in the presence of PMSF. It is activated during cell lysis and remains associated with the nuclear fraction of the lysate during subsequent washes. The isolation of intact nuclei is important for the ultimate extraction of undegraded histone, especially from sources in which the risks of autolysis are high or unknown.

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Profile of the body surface proteolytic systém in Apis mellifera quee

Czech Journal of Animal Science ◽

10.17221/150/2009-cjas ◽

2011 ◽

Vol 56 (No. 1) ◽

pp. 15-22 ◽

Cited By ~ 6

Author(s):

A.J. Strachecka ◽

M.M. Gryzińska ◽

M. Krauze ◽

K. Grzywnowicz

Keyword(s):

Protease Inhibitor ◽

Honey Bee ◽

Proteolytic Activity ◽

Body Surface ◽

Protease Activity ◽

Serine Proteases ◽

Developmental Stages ◽

Cysteine Proteases ◽

The Body

The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.

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Response of fish communities to different levels of white sucker (Catostomus commersoni) biomanipulation in five temperate lakes

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f01-137 ◽

2001 ◽

Vol 58 (10) ◽

pp. 1998-2010 ◽

Cited By ~ 8

Author(s):

Philippe Brodeur ◽

Pierre Magnan ◽

Michel Legault

Keyword(s):

Fish Species ◽

Brook Trout ◽

Salvelinus Fontinalis ◽

Fish Communities ◽

White Sucker ◽

Age At Maturity ◽

Catostomus Commersoni ◽

Mass Removal ◽

Males And Females ◽

Different Levels

The goal of this study was to evaluate the response of white sucker (Catostomus commersoni), brook trout (Salvelinus fontinalis), and other fish species to the mass removal of white sucker in five Québec (Canada) lakes. White sucker removal ranged from 14.2 kg·ha1 to 31.3 kg·ha1 3 years after mass removal. In four of the study lakes, the proportion of 2+ to 4+ white sucker increased following mass removal. Mean catch and biomass per unit of effort of 1+ brook trout increased significantly in the lakes where white sucker removal was highest. All white sucker populations experienced growth increases after mass removal, and improved brook trout growth was observed in lakes where the most intensive mass removal occurred. These growth increases led to higher mean length at maturity in white sucker females and decreases in mean age at maturity in white sucker males and brook trout males and females. Mean adjusted fecundity significantly increased in white sucker and brook trout in lakes where mass removal was most intense. The present study suggests that white sucker and brook trout exhibit compensatory responses following a reduction of intra- and inter-specific competition and that these responses are related to the intensity of mass removal.

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Effect of Plant Growth Regulators on Protease Activity in Forest Floor of Norway Spruce Stand

Forests ◽

10.3390/f12060665 ◽

2021 ◽

Vol 12 (6) ◽

pp. 665

Author(s):

Ladislav Holik ◽

Jiří Volánek ◽

Valerie Vranová

Keyword(s):

Organic Matter ◽

Proteolytic Activity ◽

Protease Activity ◽

Forest Floor ◽

Soil Microbial Activity ◽

Inhibitory Effects ◽

Chlorocholine Chloride ◽

Soil Microbial ◽

Native Soil ◽

Transformation Processes

Soil proteases are involved in organic matter transformation processes and, thus, influence ecosystem nutrient turnovers. Phytohormones, similarly to proteases, are synthesized and secreted into soil by fungi and microorganisms, and regulate plant rhizosphere activity. The aim of this study was to determine the effect of auxins, cytokinins, ethephon, and chlorocholine chloride on spruce forest floor protease activity. It was concluded that the presence of auxins stimulated native proteolytic activity, specifically synthetic auxin 2-naphthoxyacetic acid (16% increase at added quantity of 5 μg) and naturally occurring indole-3-acetic acid (18%, 5 μg). On the contrary, cytokinins, ethephon and chlorocholine chloride inhibited native soil protease activity, where ethephon (36% decrease at 50 μg) and chlorocholine chloride (34%, 100 μg) showed the highest inhibitory effects. It was concluded that negative phytohormonal effects on native proteolytic activity may slow down organic matter decomposition rates and hence complicate plant nutrition. The study enhances the understanding of rhizosphere exudate effects on soil microbial activity and soil nitrogen cycle.

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FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

Journal of Experimental Medicine ◽

10.1084/jem.113.2.359 ◽

1961 ◽

Vol 113 (2) ◽

pp. 359-380 ◽

Cited By ~ 32

Author(s):

Georges Ungar ◽

Takuso Yamura ◽

Jacqueline B. Isola ◽

Sidney Kobrin

Keyword(s):

Amino Acid ◽

Guinea Pig ◽

Proteolytic Activity ◽

Guinea Pigs ◽

Protease Activity ◽

Amino Acid Esters ◽

Protein Substrates ◽

Protease Activation ◽

Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

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Effect of a protease inhibitor on in vitro stability of intact parathyroid hormone

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1258/000456303763046166 ◽

2003 ◽

Vol 40 (2) ◽

pp. 188-190 ◽

Cited By ~ 6

Author(s):

N. R. Anderson ◽

J. Nicholas ◽

M. R. Holland ◽

R. Gama

Keyword(s):

Parathyroid Hormone ◽

Protease Inhibitor ◽

Protease Activity ◽

Intact Parathyroid Hormone ◽

In Vitro Degradation ◽

Glass Tubes ◽

Edta Plasma ◽

In Vitro Stability ◽

Baseline Sample

Background: We investigated whether increased protease activity explains the increased in vitro degradation of intact parathyroid hormone (iPTH) observed in serum when compared to EDTA plasma. Methods: Pre-dialysis blood samples for iPTH were taken from 11 patients with chronic renal failure and collected into plain glass tubes, tubes containing 200 KIU/mL aprotinin (a protease inhibitor) and EDTA tubes. All sample aliquots were separated at 20 min, 1 h, 2 h, 4 h, 8 h and 24 h post collection. Results: Over 24 h, iPTH concentrations remained unchanged in EDTA tubes. iPTH concentrations were significantly lower in both plain tubes ( P < 0·01) and aprotinin tubes ( P < 0·001) at 24 h when compared to the baseline sample (20 min). At 24 h, iPTH concentrations in EDTA tubes were higher than in plain tubes ( P < 0·001) and aprotinin tubes ( P < 0·01). The addition of aprotinin to plain tubes significantly reduced the degradation of iPTH ( P < 0·05) at 24 h. Conclusion: Aprotinin significantly reduces the in vitro degradation of iPTH in plain tubes at 24 h from 24·7% to 9·6%. We suggest that increased protease activity contributes to the decline in serum iPTH over time. As this is observed in serum and not plasma it suggests that the increased protease activity may be due to the clotting process.

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Resistance of Phlebotomus papatasi to infection with Leishmania donovani is modulated by components of the infective bloodmeal

Parasitology ◽

10.1017/s0031182098003321 ◽

1998 ◽

Vol 117 (5) ◽

pp. 467-473 ◽

Cited By ~ 28

Author(s):

Y. SCHLEIN ◽

R. L. JACOBSON

Keyword(s):

Proteolytic Activity ◽

Trypsin Inhibitor ◽

Alkaline Protease ◽

Protease Activity ◽

Leishmania Donovani ◽

Blood Feeding ◽

Growth Conditions ◽

Enzymatic Activities ◽

Phlebotomus Papatasi ◽

Proteolytic Activities

The circumstances which permit the establishment of Leishmania infections in sandflies were investigated by altering the growth conditions for L. donovani parasites in the unsuitable vector Phlebotomus papatasi. Only 5·0% of the sandflies harboured a few parasites 3 days after feeding on promastigotes in defibrinated blood. Heparinized blood or the addition of trypsin inhibitor to the meals allowed persistence of infections (day 6) in 9·9% and 25·8% of the flies respectively. Meals of erythrocytes, saline and amastigotes produced 44·4% fly infection on day 6, while similar promastigote-initiated infections remained in 70·3% of the flies. Proteolytic activities in the guts of sandflies fed on the above meals without parasites, were the highest after defibrinated bloodmeals. Erythrocytes with saline decreased the maximal alkaline protease level from 20·8 U to 13·5 U/fly; that of trypsin from 3·9 U to 1·8 U/fly and that of the aminopeptidase from 5·5 U to 3·9 U/fly. After meals of heparinized blood, the maximal alkaline protease activity (12·0 U/fly) was also much lower than after defibrinated blood-feeding. The different diets which resulted in comparatively low enzymatic activities, including blood with trypsin inhibitor, also promoted the survival of infections. This implies that the proteolytic activity in the sandfly gut modulates the vector susceptibility.

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The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth, biofilm formation and proteolytic activity in Trichosporon spp.

Biofouling ◽

10.1080/08927014.2017.1350947 ◽

2017 ◽

Vol 33 (8) ◽

pp. 640-650 ◽

Cited By ~ 9

Author(s):

Rossana de Aguiar Cordeiro ◽

Rosana Serpa ◽

Patrícia Bruna Leite Mendes ◽

Antonio José de Jesus Evangelista ◽

Ana Raquel Colares Andrade ◽

...

Keyword(s):

Protease Inhibitor ◽

Proteolytic Activity ◽

Biofilm Formation ◽

Aspartyl Protease ◽

Planktonic Growth

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Biological Procedures Online ◽

10.1186/s12575-020-00138-0 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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