Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airway

Previously we isolated a new trypsin-like enzyme designated human airway trypsin-like protease (HAT) from human sputum. In this study,we examined in vitro whether HAT was related to the prevention of fibrin deposition in the airway lumen by cleaving fibrinogen. In mucoid sputum samples from patients with chronic airway diseases, the concentration of fibrinogen, as measured by ELISA, was in the range of 2-20μg/ml, and trypsin-like activity, as measured by spectrofluorometry was in the range of 10-50 milliunits (mU)/ml. We showed by gel filtration that the trypsin-like activity of mucoid sputum was mainly due to HAT. We examined the effects of HAT on human fibrinogen at pH 7.4 and 8.6. Fibrinogen was used at concentrations of 4-2,000μg/ml and HAT purified from sputum at concentrations of 0.6-10 mU/ml. As shown by SDS-polyacrylamide gel electrophoresis, HAT cleaved fibrinogen, especially its α-chain, regardless of the concentration of fibrinogen. Pretreatment of fibrinogen with HAT resulted in a decrease or complete loss of its thrombin-induced clotting capacity, depending on the duration of pretreatment with HAT and the concentration of HAT. From these results we postulated that HAT may participate in the anticoagulation process within the airway, especially at the level of the mucous membrane, by cleaving fibrinogen transported from the blood stream

Noninvasive Demonstration of Dual Coronary Artery Fistulas to Main Pulmonary Artery with 64-Slice Multidetector-Computed Tomography: A Case Report

Coronary artery fistulas, including coronary pulmonary fistulas, are usually discovered accidently among the adult population when undergoing invasive coronary angiographies. We report here a 58-year-old woman with dual fistulas originating from the left anterior descending coronary artery and right coronary sinus to the main pulmonary artery, demonstrating noninvasively with multidetector-computed tomography (MDCT) and transthoracic echocardiography (TTE)

Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase

To clarify the pathophysiological role of cathepsins in rheumatoid arthritis (RA), we investigated whether cathepsin B or cathepsin L was increased in synovial fluid (SF) of RA joints, and whether the cathepsin isolated from SF of RA patients activated pro-urokinase or not. Thus, we estimated the content of cathepsins in SF of RA patients by measuring their activities by fluorospectrometry, using Z-Phe-Arg-MCA as the substrate. Cathepsin activity was approxymately 4-fold higher in the SF of RA patients than in those of patients with osteoarthritis. Cathepsin B and cathepsin L were separated by cation-exchange column chromatography. As a result, a large peak corresponding to cathepsin B and a very small peak correponding to cathepsin L were detected. Biochemical sequential fractionation of the cathepsin purified from the SF showed that the large peak was mainly composed of cathepsin B. This purified enzyme induced conversion of pro-urokinase to urokinase, and the Km for pro-urokinase was approximately 8.27μM. These findings indicated that an imbalance between cathepsin B and its inhibitors occurred due to increased concentrations of active cathepsin B in RA articular lesions, and that cathepsin B might be related to the degradation of cartilage in RA by activating the fibrinolytic cascade

Human airway trypsin-like protease in saliva

We first discovered human airway trypsin-like protease (HAT) in human mucoid sputum. Precursor HAT (47 kDa), a cell surface type II transmembrane serine protease, is proteolyzed to mature HAT (27 kDa). Hitherto, HAT has not been detected in other biological fluids except for human sputum. We aimed to clarify whether human saliva contains mature HAT. Trypsin-like protease was isolated from saliva of healthy volunteers by a method adopted for isolation of HAT from sputum using Boc-Phe-Ser-Arg-MCA as the substrate. Biochemical properties of purified protease were similar to those of recombinant HAT (rHAT). HAT concentration in saliva was measured by ELISA, and immunoreactive HAT : total protein ratio (ng/mg) in saliva samples from healthy subjects was similar to that in mucoid sputum. RT-PCR showed that HAT mRNA was expressed in human gingival epithelial cells but not in gingival fibroblasts. Both indirect immunofluorescence and western blotting using monoclonal antibody for α-smooth muscle actin (α-SMA ; a myofibroblast marker) showed that HAT enhanced α-SMA fiber expression in gingival fibroblasts. These results indicate that both mucoid sputum and saliva from healthy subjects have similar concentrations of mature HAT, and HAT is related to certain physiological functions and pathological states of myofibroblasts in the oral cavity

Effect of saliva collection method on the concentration of protein components in saliva

In order to clarify how we collect saliva for analyzing salivary protein in aged subjects who cannot eat well, we compared the effects of suction, spitting and the swab saliva collection method on the yield of protein components in saliva samples from normal volunteers. The saliva collected by suction, spitting and the swab method were designated as, Saliva I, II and III, respectively. The saliva volume collected by Saliva I was about 2-fold greater than that by of Saliva II and III. This is mainly due to the fact that saliva secretion was stimulated by the suction itself. The content of total protein, S-IgA, trypsin-like activity and human airway trypsin-like protease(HAT)were almost the same in Saliva I and II, and significantly lower in Saliva III than in Saliva I and II. Kallikrein activity was almost the same in Saliva I, II and III. The concentration of eachtotal protein, S-IgA, kallikrein activity, trypsin activity and HAT in Saliva I were significantly positively correlated with that in Saliva II. These results indicate that we can obtain information of change of salivary protein by analyzing saliva collected by suction method, although this method caused the stimulation of saliva to some extent

Relationships between activity of daily living, and oral cavity care and the number of oral cavity microorganisms in patients with cerebrovascular diseases

We examined the relationships among the activity of daily living (ADL), oral cavity care, and the number of oral cavity microorganisms in 40patients with cerebro-vascular diseases (CVD). The CVD patients were classified into 4groups, I, II, III and IV based on their ADL and the method used for oral cavity care. The ADL was highest in group I and lowest in group III. Only the patients of only group III could not eat by themselves and were receiving naso-esophageal feeding. Oral cavity care was performed by the patients themselves in groups I and IV, but was performed by caregivers in groups II and III. The group IV patients had no teeth, but could eat by themselves using full dentures. The numbers of microorganisms in the pharyngeal swabs from the 4groups were measured and expressed as colony-forming units (cfu). The numbers of both Staphylococci spp. and Candida spp. were significantly higher in group III than in the other groups. Moreover, Pseudomonas aeruginosa was isolated only from patients of group III (in about 66%). The oral cavity care by caregivers was almost the same in groups II and III, but the numbers of oral cavity microorganisms were significantly higher in group III than in group II. These results indicated that microorganisms grow more easily in the oral cavities of CVD patients with low ADL compared with CVD patients with higher ADL, and that eating is thought to be important for the prevention of an increase of microorganisms in the oral cavity

Effect of human airway trypsin-like protease on intracellular free Ca2+ concentration in human bronchial epithelial cells

It has been shown that human airway trypsin-like protease (HAT) is localized in human bronchial epithelial cells (HBEC), and trypsin activates protease-activated receptor-2(PAR-2). Activation of PAR-2 activates G-protein followed by an increase of intracellular free Ca2+, [Ca2+]in. This study was undertaken to clarify whether HAT can activate PAR-2in HBEC or not. RT-PCR showed that HAT mRNA is expressed in HBEC, and PAR-2 mRNA is the most strongly expressed of the known PARs in HBEC. Both PAR-2 agonist peptide (PAR-2 AP) and HAT increased [Ca2+]in in HBEC in a biphasic fashion a prompt, sharp increase (peak I) and a sustained low plateau (peak II). PAR-2 AP over 100-200 μM and HAT over 200-300 mU/ml (0.08-0.12 μM) induced both peak I and II, and PAR-2 AP below100 μM and HAT below 200 mU/ml induced only peak II. Both PAR-2 AP-induced and　HAT-induced peak I were induced by Ca2+ mobilization from intracellular stores, because　they appeared even in Ca2+-free medium. Both PAR-2 AP-induced and HAT-induced peak II were induced by an influx of extracellular Ca2+, because they were abolished in Ca2+-free medium. The Ca2+ response to HAT was desensitized by exposure of HBEC to PAR-2 AP. These results indicate that HBEC have a functional PAR-2, and HAT regulates cellular functions of HBEC via activation of PAR-2

Radon Emanation and dose estimation from artificial radon spa source

International Symposium on Natural Radiation Exposures and Low Dose Radiation Epidemiological Studie