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: Inflammation plays an important role in the postoperative morbidity of organs, which is related to the activation of pro-inflammatory and anti-inflammatory cytokines. Ulinastatin (Urinary trypsin inhibitor, UTI) is a serine protease inhibitor found in human urine or serum that inhibits the activation of human leukocyte elastase. This study examined the effect of UTI on the inflammation response in patients undergoing a gastrectomy. METHODS: Thirty patients scheduled to undergo a gastrectomy were divided into two groups as follows: Control group (untreated, n = 15) and UTI group (100,000 units of UTI were continuously injected intravenously for 2 hours, n = 15). Arterial blood was sampled before surgery (T0), 10 minutes after its onset (T1), at its end (T2), and 1 hour after surgery (T3) to measure the level of cytokines. RESULTS: Both the control and treatment groups had higher interleukin (IL)-6 levels at T2 and T3 than T0, and the level increased with time. However, the increase was smaller in the treatment group. The IL-8 levels were not activated significantly in any of the groups. CONCLUSIONS: UTI inhibits the secretion of IL-6, which is an inflammatory cytokine produced after a gastrectomy. This shows that UTI can decrease the inflammation reaction caused by surgical stress.
Cytokines ; Gastrectomy ; Glycoproteins ; Humans ; Inflammation ; Interleukin-6 ; Interleukin-8 ; Interleukins ; Leukocyte Elastase ; Serine Proteases ; Trypsin

Recently, many works to treat chronic corneal ulcer have been progressed. It has been reported that the Aprotinin, one of them, is serine protease inhibitor and is useful to treat therapy-resistant chronic corneal ulcer because it decreases the plasmin level in tear fluid that was increased in corneal ulcer. We performed this study to evaluate the effect of Aprotinin to the reepithelization of cornea according to its concentration. We made corneal burn in rabbits and instilled topical antibiotics and Aprotinin 500u/ml and 200u/ml, four times a day. After instillation, we compared the process of corneal epithelial wound healing, according to the time interval, clinically and histopathologically in each group. As a result, wound healing of cornea treated with combination of antibiotics and Aprotinin was delayed rather than that treated with antibiotics only. And combination therapy with Aprotinin 500n/ml is more effective than with Aprotinin 2000n/ml. This data suggests that high concentration of Aprotinin alone is not helpful to tresat the therapyresistant chronic corneal ulcer.
Anti-Bacterial Agents ; Aprotinin* ; Burns* ; Cornea ; Corneal Ulcer ; Fibrinolysin ; Rabbits* ; Serine Proteases ; Wound Healing

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

Uremic pruritus is a common problem in patients with end-stage renal disease (ESRD), but the underlying mechanisms are not yet fully understood. We aimed to investigate the association between severity of uremic pruritus and cutaneous serine protease activity, as well as proteinase-activated receptor-2 (PAR-2) expression. Twelve ESRD patients with pruritus, 4 ESRD patients without pruritus, and 6 healthy controls were enrolled. Skin biopsies were obtained from the abdomen. Protease activity and PAR-2 expression in the epidermis were examined by in situ zymography and confocal laser microscopy, respectively. All ESRD patients presented more pronounced cutaneous protease activity compared with that in healthy controls. The skin samples from the patients with pruritus showed higher protease activity than either nonpruritic ESRD patients or healthy controls. The epidermis in all samples of ESRD patients presented higher immunoreactivity against PAR-2 versus those of healthy controls. In addition, correlation analysis between PAR-2 expression and VAS pruritus scores showed a significant positive correlation. Our data suggests that levels of serine protease and PAR-2 expression could play important roles in the pathogenesis of uremic pruritus.
Abdomen ; Biopsy ; Epidermis ; Humans ; Kidney Failure, Chronic* ; Microscopy, Confocal ; Pilot Projects* ; Pruritus* ; Serine Proteases ; Skin ; Uremia

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