Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Acanthamoeba castellanii ; Acanthamoeba ; Cathepsin L ; Cathepsins ; Cysteine Proteases ; Cysteine ; Fibronectins ; Genes, vif ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; Sequence Analysis ; Trophozoites ; Virulence

OBJECTIVETo observe the effect of Wenyang Huoxue Lishui Recipe (WHLR) containing serum on the expression of cathepsin L (CatL) in puromycin aminonucleoside-induced injury of mouse glomerular podocytes.

METHODSMouse podocyte cells (MPCs) in vitro cultured were divided into the normal control group, the model group, the dexamethasone (DEX) group, 10% WHLR containing serum group, 20% WHLR containing serum group, the vehicle serum control group. MPCs in the normal control group were cultured at 37 degrees C culture solution for 24 h. 45 mg/L puromycin was acted on MPCs in the model group for 24 h. On the basis of puromycin intervention, 1 limol/L DEX was co-incubated in MPCs of the DEX group for 24 h; 10% or 20% WHLR containing serum was co-incubated in MPCs of the 10% WHLR containing serum group and 20% WHLR containing serum group for 24 h. The vehicle serum control group was also set up by incubating with WHLR containing serum alone for 24 h. The expression of CatL and its substrate Synaptopodin in podocytes were detected by cell immunofluorescence staining. FITC-conjugated phalloidin was used to stain F-actin. A cortical F-actin score index (CFS index) was designed to quantify the degree of cytoskeletal reorganization in cultured podocytes.

RESULTSCompared with the normal control group, the expression of synaptopodin significantly decreased and the expression of CatL significantly-increased in the model group. F-actin arranged in disorder, gradually forming pericellular F-actin ring. CFS index was obviously elevated (P < 0.01). Compared with the model group, the epression of synaptopodin increased, the expression of CatL decreased, and CFS index also decreased in the DEX group, 10% WHLR containing serum group, and 20% WHLR containing serum group (P < 0.05, P < 0.01). Compared with the DEX group, the expression of synaptopodin decreased in 10% WHLR containing serum group, CFS index also decreased in 20% WHLR containing serum group (P < 0.05).

CONCLUSIONSWHLR could up-regulate the expression of synaptopodin, down-regulate the expression of CatL, and alleviate cytoskeletal reorganization of F-actin. It was helpful to stabilize the cytoskeleton of F-actin and improve the merging of podocytes.

Actins ; metabolism ; Animals ; Cathepsin L ; metabolism ; Cells, Cultured ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Kidney Glomerulus ; cytology ; Mice ; Microfilament Proteins ; metabolism ; Podocytes ; drug effects ; pathology ; Puromycin Aminonucleoside ; adverse effects ; Up-Regulation

BACKGROUNDCathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).

METHODSPrimary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance.

RESULTSUVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.

CONCLUSIONSUVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These findings provide a new possible molecular approach for antiphotoaging therapy.

Anthracenes ; pharmacology ; Cathepsin L ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibroblasts ; cytology ; drug effects ; metabolism ; radiation effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; radiation effects ; Oncogene Proteins v-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Pyridines ; pharmacology ; Skin ; cytology ; Ultraviolet Rays

OBJECTIVE: To test cathepsin L as a biomarker of myocardial ischemia by examination of cathepsin L expression in plasma after myocardial ischemia and ischemia-reperfusion in rats. METHODS: The rat models were established and divided in acute myocardial ischemia model (myocardial ischemia 30 min, 1 h, 2 h groups), ischemia-reperfusion model (ischemia-reperfusion group), and isoflurane-pretreated ischemia-reperfusion model (isoflurane-pretreated group), respectively. Normal control group and sham-operated group were established as contrast. The contents of cathepsin L in plasma were examined by ELISA and myocardial infarction areas were measured after TTC staining. RESULTS: No statistical significant changes were found among the experimental groups compared with the normal control group and sham-operated group (P>0.05). The cathepsin L from the ischemia-reperfusion group increased to 2.37 times compared with the normal control group (P<0.05). The cathepsin L and myocardium infarction size of isoflurane-pretreated group decreased compared with the ischemia-reperfusion group (P<0.05). CONCLUSION The cathepsin L in plasma is not a promising biomarker of acute myocardial ischemia. Isoflurane preconditioning can reduce the cathepsin L in plasma caused by ischemia-reperfusion injury.
Animals ; Biomarkers/blood* ; Cathepsin L/analysis* ; Isoflurane ; Myocardial Infarction/metabolism* ; Myocardial Ischemia ; Myocardial Reperfusion Injury/metabolism* ; Myocardium ; Rats

PURPOSE: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. MATERIALS AND METHODS: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. RESULTS: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. CONCLUSION: Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.
Animals ; Cathepsin B/*genetics/metabolism ; *Down-Regulation ; Gene Expression Regulation ; Hyperoxia/*genetics ; Interleukin-10/*pharmacology/physiology ; L-Lactate Dehydrogenase/metabolism ; Necrosis/chemically induced ; Oxygen/metabolism ; Rats

BACKGROUND: Muscle wasting in sepsis is associated with increased proteolysis. Interleukin-15 (IL-15) has been characterized as an anabolic factor for skeletal muscles. Our study aims to investigate the role of IL-15 in sepsis-induced muscle atrophy and proteolysis. METHODS: Mice were rendered septic either by cecal ligation and puncture or by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg i.p.). Expression of IL-15 mRNA and protein was determined by reverse transcriptase polymerase chain reaction and Western blot analysis in the control and septic limb muscles. C2C12 skeletal muscle cells were stimulated in vitro with either LPS or dexamethasone in the presence and absence of IL-15 and sampled at different time intervals (24, 48, or 72 hours). IL-15 (10microg/kg) was intraperitoneally administered 6 hours before sepsis induction and limb muscles were sampled after 24 hours of sepsis. Cathepsin L activity was determined to measure muscle proteolysis. Atrogin-1 and muscle-specific ring finger protein 1 (MuRF1) expressions in limb muscle protein lysates was analyzed. RESULTS: IL-15 mRNA expression was significantly lower in the limb muscles of septic mice compared to that of controls. Cathepsin L activity in C2C12 cells was significantly lower in presence of IL-15, when compared to that observed with individual treatments of LPS or dexamethasone or tumor necrosis factor alpha. Further, the limb muscles of mice pre-treated with IL-15 prior to sepsis induction showed a lower expression of atrogin-1 and MuRF1 than those not pre-treated. CONCLUSION: IL-15 may play a role in protection against sepsis-induced muscle wasting; thereby, serving as a potential therapeutic target for sepsis-induced skeletal muscle wasting and proteolysis.
Animals ; Atrophy ; Blotting, Western ; Cathepsin L ; Dexamethasone ; Extremities ; Fingers ; Injections, Intraperitoneal ; Interleukin-15 ; Ligation ; Mice ; Muscle Proteins ; Muscle, Skeletal ; Muscles ; Muscular Atrophy ; Proteolysis ; Punctures ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Sepsis ; Tumor Necrosis Factor-alpha

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.
Animals ; Caspase 3 ; Cathepsin B/*metabolism ; Cell Death ; Cell Hypoxia ; Enzyme Activation ; Female ; In Situ Nick-End Labeling ; L-Lactate Dehydrogenase/analysis ; Lung/metabolism ; Necrosis/metabolism ; Oxygen ; Pneumocytes/cytology/*metabolism ; Polymerase Chain Reaction ; Pregnancy ; Pulmonary Alveoli/cytology/embryology/*enzymology ; Rats ; Rats, Sprague-Dawley

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Antibodies, Neutralizing/immunology ; Cathepsin L/genetics/*metabolism ; *Cell Movement ; Cells, Cultured ; Comet Assay ; Dependovirus/genetics ; Endothelial Cells/cytology/*metabolism ; Fibroblast Growth Factor 2/genetics/immunology/*metabolism ; Gene Transfer Techniques ; Humans ; Immunoblotting ; JNK Mitogen-Activated Protein Kinases ; Lac Operon/genetics ; Mass Spectrometry ; Matrix Metalloproteinase 1/biosynthesis/genetics ; Muscle, Skeletal/*metabolism ; Neovascularization, Physiologic ; Plasminogen Activator Inhibitor 1/biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.
Amino Acid Sequence ; Animals ; Cathepsin L ; genetics ; Cloning, Molecular ; Cysteine Proteases ; genetics ; Genes, Helminth ; genetics ; Molecular Sequence Data ; Nematoda ; enzymology ; genetics ; Phylogeny ; Sequence Alignment ; Sequence Analysis, Protein ; Sequence Homology, Amino Acid ; Solanum tuberosum ; parasitology

OBJECTIVETo study the effect of Bushen Tiaojing Recipe (BTR) and Xiaoyao Pill (XP) on cathepsin-L (Cat-L) mRNA in mice.

METHODSImmature mice were randomly divided into the normal group, the control group, the BTR group and the XP group, three in each group. Cat-L mRNA expression in mice was detected using reverse transcription polymerase chain reaction (RT-PCR) at 0, 4, 8 and 12 h after injecting 5 IU (human chorionic gonadotropin, HCG).

RESULTSCat-L mRNA expression increased gradually after HCG injection, the relative levels in the control group at 0, 4, 8 and 12 h were 0.066 +/- 0.005, 0.383 +/- 0.045, 0.737 +/- 0.024 and 1.036 +/- 0.073 respectively, comparisons between different time-points showed significant difference (P < 0.01). Compared with the control group, the Cat L mRNA expression was higher at 4 h in both BTR and XP groups (P < 0.01), at 8 h in the XP group (P < 0.05), and at 12 h in BTR group after injecting HCG (P < 0.05). Compared with the control group, cat L mRNA expression showed no statistic difference at 8 h in BTR group and at 12 h in XC group.

CONCLUSIONSBTR promoted the ovulation by enhancing the expression of CatL gene, and that of XP by advancing the peak of CatL gene expression.

Animals ; Cathepsin L ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gonadotropins ; administration & dosage ; Humans ; Mice ; Mice, Inbred Strains ; Ovulation ; drug effects ; RNA, Messenger ; genetics