Tissue-type plasminogen activator (tPA) is a serine proteinase which plays important roles in functional and structural synaptic plasticity, neural migration, as well as excitotoxic injuries in several pathological situations including ischemic stroke, seizure and Alzheimer's disease (AD). It has been suggested that a divalent cation zinc also plays pathological roles in ischemia and seizure. Interestingly, it has been suggested that zinc and tPA may negatively regulate the activity or the level of each other by mechanism involving physical interaction between the two. In the present study, we investigated the effect of zinc in tPA activity and expression in rat primary astrocyte. Astrocytes were transiently exposed to 20~200micrometer Zn2+ for 2 h and then were recovered for 24 h. In the culture supernatants, zinc treatment concentration-dependently inhibited the activity of tPA which was determined by casein-plasminogen zymography. There was only marginal changes, if any, in the level of tPA mRNA and protein. On the other hand, the activity of an endogenous inhibitor of tPA, plasminogen activator inhibitor-1 (PAI-1) as well as its expression was increased by zinc treatment in a concentration-dependant manner. These results suggest that zinc-induced decrease in tPA activity was also, at least in part, regulated by indirect way by regulating the level of PAI-1. The decrease in tPA activity may be a part of body's plan to reduce excitotoxic neural injury in a condition of elevated zinc in the brain.
Alzheimer Disease ; Animals ; Astrocytes ; Brain ; Hand ; Ischemia ; Plasminogen ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Plastics ; Rats ; RNA, Messenger ; Seizures ; Serine Proteases ; Stroke ; Tissue Plasminogen Activator ; Zinc

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with V_max of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of K_cat/K_m of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 ^oC. Zn²⁺ promoted the enzymatic activity while Ca²⁺, Mg²⁺, Na⁺, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.
Animals ; Cattle ; Pancreatic Elastase/genetics* ; Elastin/genetics* ; Tandem Mass Spectrometry ; Serine Proteases/genetics*

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.
Animals ; Astrocytes* ; Brain ; Cell Survival ; Humans ; Neurons ; p38 Mitogen-Activated Protein Kinases ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators* ; Plasminogen* ; Plastics ; Proteasome Endopeptidase Complex ; Proteasome Inhibitors ; Rats* ; RNA, Messenger ; Serine Proteases ; Tissue Plasminogen Activator ; Ubiquitin ; Up-Regulation*

OBJECTIVE: The most important biologic characters of malignant tumor are invasion and metastasis, and extracellular matrix is first barrier in metastatic process. Therefore, proteases are linked to the malignant phenotype of different solid tumor. METHODS: In this study, the expression of the matrix metalloproteinase (MMP)-2 and MMP-9 and of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) in the ovarian epithelial tumors was investigated. Immunohistochemical expression of MMP-2, MMP-9, uPA, and PAI-1 were analyzed in formalin fixed tumor tissues of 20 benign cystadenomas, 20 low malignant potential (LMP) tumors, and 20 malignant ovarian cancer, including 10 FIGO stage I cancer and 10 stage III cancer. In the same tissue extracts, DNA levels of MMP-2, MMP-9, uPA, and PAI-1 were determined by PCR. RESULTS: The immunohistochemical expression and DNA level of MMP-2, MMP-9, uPA, and PAI-1 were low in benign ovarian tumors but significantly increased LMP tumors and malignant ovarian cancers. The highest values of all of the proteolytic enzymes were detected in stage III ovarian cancers with omentum metastases. There are significant correlations between expression of uPA and MMP. CONCLUSION: These results suggest that high expression of MMP-2, MMP-9, and uPA is associated with activity of tumor invasion and metastasis, and expressions of MMP-2 and MMP-9 are correlated with uPA activity.
Cystadenoma ; DNA ; Extracellular Matrix ; Female ; Formaldehyde ; Neoplasm Metastasis ; Omentum ; Ovarian Neoplasms ; Ovary* ; Peptide Hydrolases ; Phenotype ; Plasminogen Activator Inhibitor 1 ; Plasminogen Activators ; Polymerase Chain Reaction ; Serine Proteases ; Tissue Extracts ; Urokinase-Type Plasminogen Activator*

Aprotinin is a serine protease inhibitor and a powerful antifibrinolytic agent, derived from the inhibition of plasmin and kallikrein. Therefore, it is widely used in cardiopulmonary bypass surgery or major surgery for reducing bleeding and blood transfusion requirements. Aprotinin is rapidly eliminated from the circulation by glomerular filtration and is actively reabsorbed in the renal tubular system, where it is stored, metabolized, and eliminated over the following 5-6 days. Because of this metabolism, concerns have been raised regarding the possibility that aprotinin may impair renal function due to a toxic effects on proximal tubular cells. We report three cases of postoperative renal failure after aprotinin had been used during surgery. Two patients, Jehovah's Witnesses who refused blood transfusion, required hemodialysis. One patient, who underwent spinal orthopedic surgery, was administered aprotinin to reduce intraoperative blood loss, and developed acute renal dysfunction. The patient recovered after supportive therapy.
Acute Kidney Injury* ; Aprotinin* ; Blood Transfusion ; Cardiopulmonary Bypass ; Fibrinolysin ; Filtration ; Hemorrhage ; Humans ; Jehovah's Witnesses ; Kallikreins ; Metabolism ; Orthopedics ; Renal Dialysis ; Renal Insufficiency ; Serine Proteases

10–30% of patients with pancreatitis can be categorized as idiopathic pancreatitis, and some of them may be due to genetic alterations. Since hereditary pancreatitis develops from pediatric patients with symptoms related to pancreatitis, which usually progresses to chronic pancreatitis around 30 years of age, special attention should be paid to the development of pancreatic cancer in such patients. Up to now, there have been more than 30 genetic alterations associated with pancreatitis. Alterations in protease serine 1 (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR) and chymotrypsin C (CTRC) are common, which show diversity according to race and region. It is important to understand the characteristics of Korean patients with idiopathic pancreatitis through genetic studies. The purpose of this article is to review the role of genetic variations in the pathophysiology of idiopathic pancreatitis and to survey the results of Korean studies of idiopathic pancreatitis.
Chymotrypsin ; Continental Population Groups ; Cystic Fibrosis Transmembrane Conductance Regulator ; Genetic Variation ; Humans ; Pancreatic Neoplasms ; Pancreatitis* ; Pancreatitis, Chronic ; Serine ; Serine Proteases

No abstract available.
Korea ; Prekallikrein

BACKGROUND: Elafin is a serine proteinase inhibitor first discovered in keratinocytes from psoriatic epidermis. The molecular structure of elafin contains two different functional domains; one for the proteinase inhibitor, which is directed against elastase and proteinase-3, and the other for transglutaminase substrate. As this unique structural characteristic implies, elafin would be expected to have two distinct biologic functions in tissues. But, the roles and biological features of elafin have not been extensively studied in the skin. OBJECTIVES: This study was designed to demonstrate the immunohistochemical localization of elafin in inflammatory and keratinizing skin disorders, and to elucidate its biological functions. MATERIALS AND METHODS: The pre-elafin sequence was amplified by PCR using a human epidermal cDNA library and expression construct which was ligated into an expression vector. Then, the expressed proelafin sequence was purified and injected intradermally into rabbits to raise a polyclonal antibody. The skin biopsy samples of various skin diseases and normal controls were used for immunohistochemical staining to detect elafin expression. RESULTS: 1)Expression of elafin was observed in infected epidermis, and is believed to be involved in the defense mechanism of the skin. 2)In dermal and subcutaneous inflammatory diseases, epidermal elafin expression was influenced by the location of the inflammation. 3)Elafin was expressed in bullous dermatoses accompanied by acantholysis or spongiosis of epidermal cells. 4)Expression of elafin was upregulated in papulosquamous skin diseases which was characterized by epidermal hyperplasia and abnormal differentiation. 5)Elafin was expressed in keratinizing skin diseases that was accompanied by abnormal differentiation of epidermal cells. 6)Expression of elafin was demonstrated in skin tumors that showed proliferation of suprabasal cells. The intensity of expression is not related to the degree of malignancy, but to the degree of differentiation of tumor cells. CONCLUSION: Induction of elafin expression may play an important role in protecting the skin components against tissue damage. Elafin expression is related to the abnormal proliferation and differentiation of suprabasal cells. This means that elafin may be used as a marker of these conditions.
Acantholysis ; Biopsy ; Elafin* ; Epidermis ; Gene Library ; Humans ; Hyperplasia ; Immunohistochemistry ; Inflammation ; Keratinocytes ; Molecular Structure ; Pancreatic Elastase ; Polymerase Chain Reaction ; Rabbits ; Serine Proteases ; Skin Diseases ; Skin Diseases, Papulosquamous ; Skin Diseases, Vesiculobullous ; Skin*

No abstract available.
Bernard-Soulier Syndrome* ; Prekallikrein*

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