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*

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (K
Amino Acid Sequence ; Animals ; Leeches/chemistry* ; Pancreatic Elastase/antagonists & inhibitors* ; Protease Inhibitors/pharmacology* ; Proteins

Kawasaki disease is a form of vasculitis, mainly in small and medium arteries of unknown origin, occurring frequently in childhood. It is the leading form of childhood-onset acquired heart disease in developed countries and leads to complications of coronary artery aneurysms in approximately 25% of cases if left untreated. Although more than half a century has passed since Professor Tomisaku Kawasaki's first report in 1957, the cause is not yet clear. Currently, intravenous immunoglobulin therapy has been established as the standard treatment for Kawasaki disease. Various treatment strategies are still being studied under the slogan, “Ending powerful inflammation in the acute phase as early as possible and minimizing the incidence of coronary artery lesions,” as the goal of acute phase treatments for Kawasaki disease. Currently, in addition to immunoglobulin therapy, steroid therapy, therapy using infliximab, biological products, suppression of elastase secretion inside and outside the neutrophils, inactivated ulinastatin therapy and cyclosporine therapy, plasma exchange, etc. are performed. This chapter outlines the history and transition of the acute phase treatment for Kawasaki disease.
Aneurysm ; Arteries ; Biological Products ; Coronary Vessels ; Cyclosporine ; Developed Countries ; Heart Diseases ; Immunization, Passive ; Incidence ; Inflammation ; Infliximab ; Mucocutaneous Lymph Node Syndrome ; Neutrophils ; Pancreatic Elastase ; Plasma Exchange ; Prednisolone ; Vasculitis

OBJECTIVE: To establish and evaluate the model of chronic obstructive pulmonary disease (COPD) with osteoporosis induced by elastase in mice. METHODS: Twenty four healthy female 8-week-old C57BL / 6 mice (weighing about 18 g) were randomly divided into three groups. The control group was given intratracheal drip of normal saline, the experimental group 1 and the experimental group 2 were given intratracheal drip of elastase, the control group and the experimental group 1 were kept for 8 weeks and then killed, the experimental group 2 was kept for 12 weeks and then killed. HE staining was used to evaluate the histopathological changes of lung and tibia in the control and experimental groups. The levels of serum inflammatory factors and broncho alveolar lavage factors (BALF) were detected by ELISA. Micro CT was used to detect the bone mass related parameters of mouse femur. The expression of osteoclastic and osteogenic genes was detected by real-time fluorescence quantitative PCR. RESULTS: Lung histopathology showed that the structure of alveoli in the experimental group was disordered, the walls of alveoli became thin or broken, and the alveoli cavity expanded. IL-6 and TNF-α in BALF were significantly higher than those in control group (<0.001), while IL-1β and TNF-α in serum inflammatory factors were significantly higher than those in control group (<0.001). BV / TV(bone volume fraction), TB.Th(average bone trabecular thickness) and TB.N(average bone trabecular number) in the experimental group were significantly lower than those in the control group (<0.05), TB.Sp (average bone trabecular separation) and BS / BV (bone surface area fraction) in the experimental group were significantly higher than those in the control group (<0.01). Compared with the control group, the expression of osteoclast related marker genes increased in the experimental group (<0.05), but decreased in the experimental group(<0.05). The results of experiment 1 and experiment 2 were time-dependent. CONCLUSION In this study, elastase was used to construct a COPD model with osteoporosis successfully, which provides a suitable animal model for the future study of the pathogenesis of COPD with osteoporosis.
Animals ; Bone Density ; Female ; Mice ; Mice, Inbred C57BL ; Osteoporosis ; etiology ; Pancreatic Elastase ; Pulmonary Disease, Chronic Obstructive ; complications

PURPOSE: The aim of this study was to evaluate the clinical significance of inflammatory biomarkers in acute infectious diarrhea among children. METHODS: Clinical parameters including fever, bacterial and viral etiology based on stool culture and multiplex polymerase chain reaction, and nine biomarkers including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and leukocytes in blood and calprotectin, lactoferrin, myeloperoxidase, polymorphonuclear elastase, leukocytes, and occult blood in feces were evaluated in children who were hospitalized due to acute diarrhea without underlying disease. RESULTS: A total of 62 patients were included. Among these patients, 33 had fever, 18 showed bacterial infections, and 40 patients were infected with 43 viruses. Of all the biomarkers, CRP was significantly correlated with fever (p<0.001). CRP, ESR, calprotectin, lactoferrin, myeloperoxidase, fecal leukocytes, and occult blood were significantly associated with infection with bacterial pathogens (p<0.001, p=0.04, p=0.03, p=0.003, p=0.02, p=0.03, p=0.002, respectively). The combination of CRP and fecal lactoferrin at their best cut-off values (13.7 mg/L and 22.8 µg/mL, respectively) yielded a sensitivity of 72.2%, and a specificity of 95.5% for bacterial etiology compared with their individual use. CONCLUSION: Blood CRP is a useful diagnostic marker for both fever and bacterial etiology in acute pediatric diarrhea. The combination of CRP and fecal lactoferrin yields better diagnostic capability for bacterial etiology than their use alone for acute diarrhea in children without underlying gastrointestinal disease.
Bacterial Infections ; Biomarkers ; Blood Sedimentation ; C-Reactive Protein ; Child ; Diarrhea ; Feces ; Fever ; Gastrointestinal Diseases ; Humans ; Lactoferrin ; Leukocyte L1 Antigen Complex ; Leukocytes ; Multiplex Polymerase Chain Reaction ; Occult Blood ; Pancreatic Elastase ; Peroxidase ; Sensitivity and Specificity

Placenta is a special organ that contains many nutrients such as growth factors, minerals, and bioactive peptides. Dipeptides of glycine and leucine are major components of porcine placenta extracts (PPE) that has been used as an alternative of human placenta extracts. In this study, we investigated whether major peptides of PPE, Glycyl-L-Leucine (Gly-Leu), L-Leucyl-Glycine (Leu-Gly), and L-Leucyl-L-Leucine (Leu-Leu), affect skin hydration and elasticity in vitro and in vivo. We found that Gly-Leu and Leu-Gly dipeptides induced the expression of transglutaminase 1 in normal human epidermal keratinocytes (NHEKs) whereas Leu-Leu dipeptides did not. Treatment with Gly-Leu or Leu-Gly significantly increased hyaluronan (HA) synthesis in NHEKs and the upregulation of hyaluronan synthase 2 (HAS2) mRNA level was confirmed. In addition, elastase activity was inhibited in NHEKs treated with Gly-Leu or Leu-Gly dipeptides. Oral administration of Gly-Leu or Leu-Gly dipeptides increased skin hydration and elasticity in UVB-irradiated hairless mice. The significant upregulation of HA in UVB-irradiated hairless mice was observed in response to oral administration of Gly-Leu or Leu-Gly. These results suggest that the major dipeptides of porcine placenta, Gly-Leu and Leu-Gly, are potentially active ingredients for skin moisturization formulations.
Administration, Oral* ; Animals ; Dipeptides* ; Elasticity* ; Glycine* ; Humans ; Hyaluronic Acid ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Keratinocytes ; Leucine* ; Mice ; Mice, Hairless* ; Minerals ; Miners ; Pancreatic Elastase ; Peptides ; Placenta ; RNA, Messenger ; Skin* ; Up-Regulation

OBJECTIVETo analyze the clinical characteristics and causative mutation in an ethnic Han Chinese family affected with punctate palmoplantar keratoderma (PPPK).

METHODSClinical characteristics and inheritance pattern of the family were analyzed. Two seriously affected individuals from the family were investigated by whole exome sequencing. Three healthy individuals from the family and 120 non-PPPK individuals were evaluated to validate the result.

RESULTSThe family was characterized by keratotic papules on the palms and soles, which gradually increased in size and number with age and coalesced with each other, particularly over the pressure part of the palms and soles. The family has featured autosomal dominant inheritance. A heterozygous frameshift variant c.419delC in exons of the CELA1 gene was identified in all affected individuals but not among non-affected members.

CONCLUSIONA heterozygous frameshift variant c.419delC in CELA1 gene probably underlies the disease in the family affected with PPPK.

Adult ; Base Sequence ; Female ; Frameshift Mutation ; Heterozygote ; Humans ; Keratoderma, Palmoplantar ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Pancreatic Elastase ; genetics ; Pedigree ; Young Adult

Skin aging is a complex biological process due to intrinsic and extrinsic factors. Free radical oxidative is one of extrinsic factors that induce activation of collagenase, elastase and hyaluronidase. Natural product from plants has been used as antioxidant and antiaging. This study aimed to evaluate antioxidant and antiaging properties of Hibiscus sabdariffa extract (HSE) and its compounds including myricetin, ascorbic acid, and β carotene. The phytochemical of H. sabdariffa was determined using modified Farnsworth method and presence of phenols, flavonoids and tannins were in moderate content, whereas triterpenoids and alkaloids were in low content. Total phenolic content performed using Folin-Ciocalteu method, was 23.85 µg GAE/mg. Quantitative analysis of myricetin, β-carotene, and ascorbic acid of HSE was performed with Ultra-High Performance Liquid Chromatography (UHPLC) that shows 78.23 µg/mg myricetin, 0.034 µg/mg β-carotene, whilst ascorbic acid was not detected. HSE has lower activity on DPPH (IC₅₀ = 195.73 µg/mL) compared to β-carotene, the lowest in ABTS assay (IC50 = 74.58 µg/mL) and low activity in FRAP assay (46.24 µM Fe(II)/µg) compared to myricetin, β-carotene. Antiaging was measured through inhibitory activity of collagenase, elastase, and hyaluronidase. HSE had weakest collagenase inhibitory activity (IC₅₀= 750.33 µg/mL), elastase inhibitory activity (103.83 µg/mL), hyaluronidase inhibitory activity (IC₅₀ = 619.43 µg/mL) compared to myricetin, β-carotene, and ascorbic acid. HSE contain higher myricetin compared to β-carotene. HSE has moderate antioxidants and lowest antiaging activities. Myricetin is the most active both antioxidant and antiaging activities.
Alkaloids ; Antioxidants ; Ascorbic Acid ; Biological Processes ; Carotenoids ; Chromatography, Liquid ; Collagenases ; Flavonoids ; Hibiscus* ; Hyaluronoglucosaminidase ; Methods ; Pancreatic Elastase ; Phenol ; Phenols ; Skin Aging ; Tannins

BACKGROUND/AIMS: Diesel exhaust particles (DEPs) lead to elevation of reactive oxygen species, which can activate the nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain 3 (NLRP3)-inf lammasome. In this study, we elucidated whether NLRP3 -inf lammasome is activated by DEPs and whether antioxidants (N-acetylcysteine [NAC]) could inhibit such activation. METHODS: RAW 264.7 cells and ex vivo lung tissues explants obtained from elastase-induced emphysema animal models were stimulated with cigarette smoking extract (CSE), DEPs, and lipopolysaccharide, and levels of interleukin-1β (IL-1β), caspase-1 and nucleotide-binding oligomerization domain-like receptor (NLR) family members containing the pyrin domain (NLRP3)-inflammasome were assessed by Western blotting and immunohistochemistry. RESULTS: NAC and caspase-1 inhibitor suppressed CSE- and DEP-induced secretion of IL-1β in RAW 264.7 cells. The expression levels of the NLRP3-inflammasome and caspase-1 were upregulated in RAW 264.7 cells by stimulation with CSE and DEPs and were inhibited by NAC. CSE and DEPs increased the secretion of IL-1β in lung tissues from both the normal and elastase-induced emphysema groups. The secretion of IL-1β by CSE and DEPs was increased in the elastin-induced emphysema group more than that in the normal group (CSE: 309 ± 19 pg/mL vs. 151 ± 13 pg/mL, respectively, p < 0.05; DEP: 350 ± 24 pg/mL vs. 281 ± 15 pg/mL, respectively, p < 0.05). NAC inhibited CSE- and DEP-induced IL-1β secretion in both the normal and elastase-induced emphysema groups. NLRP3-inflammasome expression as determined by immunohistochemistry was increased by CSE and DEPs in both the normal and elastin-induced emphysema groups, and was suppressed by NAC. CONCLUSIONS: The NLRP3-inf lammasome is activated by DEPs in ex vivo tissue explants from elastase-induced emphysema animal model, and this activation is inhibited by NAC.
Antioxidants ; Blotting, Western ; Emphysema* ; Humans ; Immunohistochemistry ; Lung* ; Models, Animal ; Pancreatic Elastase ; Pulmonary Disease, Chronic Obstructive ; RAW 264.7 Cells* ; Reactive Oxygen Species ; Smoking ; Vehicle Emissions*

OBJECTIVE: We aimed to evaluate the efficacy and safety of a newly developed, partially retrievable flow-diverter (the FloWise) in an elastase-induced rabbit aneurysm model. MATERIALS AND METHODS: We developed a partially retrievable flow diverter composed of 48 strands of Nitinol and platinum wire. The FloWise is compatible with any microcatheter of 0.027-inch inner diameter, and is retrievable up to 70% deployment. The efficacy and safety of the FloWise were evaluated in the elastase-induced rabbit aneurysm model. The rate of technical success (full coverage of aneurysm neck) and assessment of aneurysm occlusion and stent patency was conducted by angiograms and histologic examinations at the 1-month, 3-month, and 6-month follow-up. The patency of small arterial branches (intercostal or lumbar arteries) covered by the FloWise were also assessed in the 5 subjects. RESULTS: We attempted FloWise insertion in a total of 32 aneurysm models. FloWise placement was successful in 31 subjects (96.9%). Two stents (6.2%) were occluded at the 3-month follow-up, but there was no evidence of in-stent stenosis in other subjects. All stented aneurysms showed progressive occlusion: grade I (complete aneurysm occlusion) in 44.4% and grade II (aneurysm occlusion > 90%) in 55.6% at 1 month; grade I in 90% and II in 10% at 3 months; and grade I in 90% and II in 10% at 6 months. All small arterial branches covered by the FloWise remained patent. CONCLUSION: A newly developed, partially retrievable flow-diverter seems to be a safe and effective tool of aneurysm occlusion, as evaluated in the rabbit aneurysm model.
Alloys ; Aneurysm/*chemically induced/radiography/*surgery ; Angiography ; Animals ; Arteries/pathology/surgery ; Catheters ; Cerebrovascular Circulation/physiology ; Constriction, Pathologic/chemically induced/radiography/surgery ; *Disease Models, Animal ; Humans ; Male ; Pancreatic Elastase/*pharmacology ; Platinum ; *Rabbits ; Stents/*adverse effects