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

Ticks are obligate ectoparasites and vectors of arboviruses, vickettsiate, spirochetes and parasitil protozoa of humans and domestic animals. Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes, thus they can be considered as good target antigens for a tick vaccine. In the present study, we used rapid amplification of cDNA ends protocol and primers that were designed based on the consensus amino acid motifs flanking present in all papain-like cysteine proteinases, to amplify, sequence and characterize two Rhipicephalus haemaphysaloides haemaphysaloides cathepsin L-like cysteine proteinases, named as cysA and cysB. The full length of cysA is 1168bp, encoding a 332 amino acid residue polypeptide with 36.33kD predicted molecular mass; the full length of cysB is 1153bp, encoding a 335 amino acid residue polypeptide with 37.56kD predicted molecular mass. The consensus amino acid motifs flanking presence in both deduced amino acid sequences. And both genes show high sequence homology to other tick cathepsin L-like cysteine proteinase, so they were identified as members of the cysteine proteinase gene family. Expression analysis by RT-PCR revealed that cysA and cysB were expressed differently in different periods of tick development.
Amino Acid Sequence ; Animals ; Cathepsin L ; Cathepsins ; genetics ; Cloning, Molecular ; Cysteine Endopeptidases ; genetics ; Female ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Rhipicephalus ; enzymology ; Sequence Analysis

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

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

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