Aims: Thermophilic proteases are important industrial enzymes because they can be used at high temperatures in various bioprocessing schemes. The bacterial population of the Cholistan desert was explored for thermophilic proteases and their industrial applications. Methodology and results: Three bacterial isolates K1, K5 and K7 were found promising protease producers. These isolates were preliminary identified as Bacillus based on morphological characteristics and biochemical tests (positive for catalase, oxidase and citrate tests, and negative for indole and urease tests). The isolates K1, K5 and K7 were further identified as Priestia endophytica, Lysinibacillus cresolivorans and Bacillus subtilis, respectively by phylogenetic analysis. The isolates grew best at 50 °C and P. endophytica (K1), L. cresolivorans (K5) and B. subtilis (K7) produced larger zones of hydrolysis at 37 °C, 45 °C and 50 °C at pH 7, respectively. The optimum temperature where protease activity was maximum was 65 °C for P. endophytica and L. cresolivorans and 55 °C for B. subtilis, and the optimum pH was 9. Conclusion, significance and impact of study The proteases produced by these isolates were found active at high temperatures (45 °C to 85 °C) and high pH (9-12), which make them industrially important thermoalkaliphilic proteases. These proteases successfully de-haired cow’s skin and de-stained blood from cotton cloth pieces, which are rarely tested applications of these proteases.
Desert ; Proteases ; Purification ; Thermophilic bacteria

No abstract available.
Animals ; Female ; Ovary* ; Rats* ; Serine Proteases* ; Serine*

Kunitz-type serine protease inhibitors are a class of ubiquitous protease inhibitors, which play important roles in various life activities. The structures of such inhibitors are generally stable, and are usually characterized by the presence of one or several Kunitz domains in tandem, which are able to bind to serine proteases in a manner similar to substrate binding, thereby inhibiting enzyme activity. In terms of function, Kunitz-type serine protease inhibitors are involved in processes such as blood coagulation and fibrinolysis, tumor immunity, inflammation regulation, and resistance to bacterial and fungal infections. This article summarizes the advances of Kunitz-type serine protease inhibitors and provides new ideas for the development of novel Kunitz-type serine protease inhibitors.
Protease Inhibitors ; Serine Proteases ; Serine Proteinase Inhibitors

BACKGROUND: Mucus hypersecretion in the patients with airway diseases represents poor prognosis as well as discomfort. However, there is no known therapy for its effective control. One important component of mucus is mucin, a glycosylated protein, which endows mucus with viscosity. We studied whether a proteinase has a role in mucin secretion and if so, which. METHODS: (1) Inhibition of mucin secretion Group-specific proteinase inhibitors were tested to evaluate whether a proteinase belonging to a group of proteinases plays a role in mucin secretion. Phenylmethylsulfonyl fluoride(PMSF, a serine proteinase inhibitor), E-64(a cysteine proteinase inhibitor), Pepstatin(an aspartic proteinase inhibitor) and 1, 10-Phenanthroline(a metalloproteinase inhibitor) were treated into the Calu-3 cell line for 24 hours. The enzyme linked immunoabsorbant assay(ELISA) for MUC5AC was performed to evaluate the amount of mucin secretion and to compare with a control. (2) Stimulation of mucin secretion Matrix metalloproteinase-9(MMP-9), MMP-12 and TACE(TNF-alpha converting enzyme), which are known to be related with airway diseases, were used to be treated into Calu-3 for 24 hours. ELISA for MUC5AC was performed to evaluate the amount of mucin secretion and to compare with the controls. RESULTS: (1) PMSF(10(-4)M), E-64(10(-4)M), Pepstatin(10(-6)M) and 1, 10-Phenanthroline(10(-4)M) reduced the MUC5AC secretion by 1 +/- 4.9%(mean +/- standard deviation; P=1.0 compared with the control), -6 +/- 3.9%(P=0.34), -13 +/- 9.7%(P=0.34) and 41 +/- 8.2%(P=0.03), respectively. (2) The amounts of MUC5AC secretion stimulated by MMP-9(250ng/ml), MMP-12(100ng/ml) and TACE(200ng/ml) were 103 +/- 6%(P=0.39), 102 +/- 8%(P=1.0) and 107 +/- 13%(P=0.39), respectively, compared with the controls. CONCLUSION: Metalloproteinase(s) is (are) suggested to play a role in mucin secretion. It appears that metalloproteinases, other than MMP-9, MMP-12 or TACE, affect the mucin secretion in this in vitro model.
Cell Line ; Cysteine Proteases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Metalloproteases ; Mucins* ; Mucus ; Peptide Hydrolases ; Prognosis ; Serine Proteases ; Viscosity

BACKGROUND AND OBJECTIVES: Thrombin and its interaction with platelets play a pivotal role in arterial thrombus formation. Hirudin, an anticoagulant agent derived from medicinal leeches(Hirudo medicinalis), is a unique and specific thrombin inhibitor with no effect on other serine protease. We investigated the inhibitory effect of hirudin on platelet deposition in a rabbit carotid artery eversion model of acute arterial thrombosis. MATERIALS AND METHODS: The everted arterial segments were perfused with 111 Indium-labeled human platelets only(control, n=8), and a mixed solution of 111 Indium-labeled human platelets and hirudin(30, 45, 60, 90 microgram/ml, n=3, respectively). Platelet deposition was calculated by a gamma-counter and confirmed by scanning electron microscopy. RESULTS: 1) Indium-111 labeling efficiency of platelets was 87.0+/-6.6%, and the aggregation of platelets was not changed after labeling. The number of platelets perfused through each arterial segment was 4.3 +/-0.2x10(8) platelets/ml. 2) The control group showed a platelet deposition rate of 23.9+/-7.0 % and a number of platelet deposition of 9.8+/-2.5x10(8) platelets/cm2 . 3) Platelet deposition of arteries perfused with hirudin(60 microgram/ml) was significantly decreased compared with that of the control group(2.9+/-0.6 vs 9.8+/-2.5x10(8)/cm2 , p<0.05). 4) The number of deposited platelets in hirudin-perfused arteries was dose-dependently decreased(30 microgram/ml:6.7+/-1.4x10(8) /cm2 , 45 microgram/ml: 4.8+/-1.7x10(8) /cm2 , 60 microgram/ml: 2.9+/-1.8x10(8)/cm2, 90 microgram/ml:2.9+/-1.4x10(8)/cm2: p<0.05 vs. control, respectively). 5) Scanning electron microscopic examination revealed significantly reduced platelet deposition in hirudin-perfused groups compared with control group. CONCLUSION: Hirudin inhibits effectively platelet deposition and arterial thrombus formation in a rabbit carotid artery eversion model. The antiplatelet effect of hirudin in this model suggests that hirudin may be an useful antithrombotic agent therapeutically useful in the prevention of acute arterial thrombus formation.
Arteries ; Blood Platelets ; Carotid Arteries* ; Hirudins* ; Humans ; Microscopy, Electron, Scanning ; Serine Proteases ; Thrombin ; Thrombosis

The prostate-specific antigen is a serine protease which is found only in epithelial cells of prostatic origin. Benign and malignant primary, as well as metastatic prostate cells, elaborate this protein. Benign prostatic hyperplasia can be associated with elevated levels of PSA as determined by a monoclonal assay, and prostatic weight is the most important non-cancer factor variable of PSA elevation. We estimated the in vivo prostate gland volume and resected weight of prostate in 75 men using prolate ellipse formula ultrasound analysis from Jan. 1991 to June 1992. OF the men 43 (58 %) had a PSA value of 4ng/ml or less. The PSA value was greater than 4 but less than or equal to 10 in 19 men (25 %) and greater than 10 in 13 men (17 %). A total of 24 men (35 %) had a eland volume less then 25 cm3, 16% of whom had a PSA value greater than 4. Further analysis revealed that the incidence of a PSA value greater than 4 increased as the prostate volume increased (51 % for greater than or equal to 25 but less than or equal to 50 and 62.5 % for greater than 50). The incidence of a PSA Value greater than 10 increased as resected weight of prostate increased (25 % for greater than or equal to 21 but less than or equal to 30 and 75 % for greater than 30). We found a statistically significant association between prostate gland volume and resected specimen weight to the serum PSA concentration. The prostate gland volume can be a helpful adjunct in determining the significance of PSA value clinically.
Epithelial Cells ; Humans ; Incidence ; Male ; Phosmet ; Prostate* ; Prostate-Specific Antigen ; Prostatic Hyperplasia ; Serine Proteases ; Ultrasonography

When a ribosome complex is stalled during the translation elongation process in eukaryotes, the mono-ubiquitination of Rps3 has recently been shown to be critical to ribosome quality control. We have discovered that the regulatory role of Rps3 mono-ubiquitination is controlled by a deubiquitinase. We also showed that an autophagic signal appears to be coupled to the mono-ubiquitination of Rps3p through the entrance of Ubp3p into the autophagosome in yeasts. The mono-ubiquitination of the Rps3 protein is tightly modulated by reciprocal action between the Hel2p E3 ligase and the Ubp3p deubiquitinase in yeasts and the reciprocal action between the RNF123 E3 ligase and the USP10 deubiquitinase in mammalian cells. We also found that the Ubp3p/USP10 deubiquitinases critically modulate Hel2p/RNF123-mediated Rps3p mono-ubiquitination. In addition, we found that Hel2p/RNF123 and Ubp3p/USP10 appeared to be differently localized in the ribosome complex after ultraviolet irradiation. Together, our results support a model in which coordinated ubiquitination and deubiquitination activities can finely balance the level of regulatory Rps3p mono-ubiquitination in ribosome-associated quality control and autophagy processes.
Autophagy ; Eukaryota ; Quality Control* ; Ribosomes ; Ubiquitin ; Ubiquitin-Protein Ligases* ; Ubiquitin-Specific Proteases ; Ubiquitination ; Yeasts

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.
Agar ; ATP-Dependent Proteases ; Blotting, Western ; Escherichia coli ; Host-Pathogen Interactions ; Streptococcus pneumoniae* ; Streptococcus* ; Yeasts

FtsH is a membrane-bound, ATP-dependent protease involved in various cellular functions. To understand its roles in Streptococcus pneumoniae and host-pathogen interactions, we inactivated the ftsH gene of D39 strain by inserting a tetracycline-resistance (tet) gene. Several recombinants containing the tet cassette within the ftsH gene were confirmed by Western immunoblotting for the absence of pneumococcal FtsH protein that could cross-react with antiserum raised against Escherichia coli FtsH. Compared with the wild-type D39 strain, the ftsH null mutants grew slowly with encapsulation and alpha-hemolysis on blood agar plates, but failed to grow in liquid media other than Todd Hewitt yeast extract broth. Even fresh cultures of ftsH null mutants appeared gram-negative. When the incubation temperature of liquid cultures was shifted from 37degrees C to 40degrees C, the mutants gradually lysed, whereas the shift to 30degrees C abolished further growth. The mutants also exhibited increased sensitivity to salt and remarkable growth inhibition by optochin. These observations suggest that no functional FtsH protein in pneumococcal cells causes a loss of cell surface integrity, resulting in impairment of cell growth under normal and stressful conditions.
Agar ; ATP-Dependent Proteases ; Blotting, Western ; Escherichia coli ; Host-Pathogen Interactions ; Streptococcus pneumoniae* ; Streptococcus* ; Yeasts

PURPOSE:In patients with a higherrisk of bleeding, performing CVVH with heparin or saline anticoagulation is associated with increased bleeding or thrombotic risk. Nafamostat mesilate (NM), a serine proteinase inhibitor, while inhibiting various clotting factors in filter circuit, is characterized by short half life resulting in little systemic anticoagulation effect. Accordingly, we prospectively evaluated the anticoagulant effect and safety of NM in patients with a higher risk of bleeding who underwent CVVH. METHODS:Among 43 patients with high risk of bleeding [defined by (1) INR>2, aPTT>20 sec, platelet< 50,000/mm3 or (2) ongoing bleeding, major hemorrhage/surgery in the last 48 hrs], 20 patients were treated with continuous nafamostat mesilate infusion (10-20 mg/hr) and remaining 23 patients were treated with saline bolus infusion (100 mLq 1 hr) for CVVH anticoagulation. RESULTS:As compared with saline bolus group, mean circuit life was significantly longer in NM infusion group (28.73+/-12.67 versus 16.34+/-7.86, p=0.001). There was no significant bleeding complication in either saline bolus or NMinfusion group. In subgroup analysis according to the presence of abnormal coagulation status (defined by INR>2, aPTT>20 sec, platelet<50,000/mm3), the positive effect of NM on circuit lifespan persisted irrespective of the coagulation status. CONCLUSION:As compared with saline bolus, nafamostat mesilate infusion was associated with higher CVVH filter life. In patients with high risk of bleeding, nafamostat mesilate can be used as a safe and effective anticoagulant for CVVH with acceptable filter life
Guanidines ; Half-Life ; Hemofiltration ; Hemorrhage ; Heparin ; Humans ; Mesylates ; Prospective Studies ; Serine Proteases