Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

[Cathepsin-B involved in effect of electroacupuncture by inhibiting the activation of NLRP3 inflammasome in rats with acute gouty arthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on Cathepsin-B in the synovium of the knee joint of acute gouty arthritis(AGA) rats, so as to explore the mechanism of EA in the treatment of AGA. METHODS: A total of 60 male Wistar rats were randomly divided into normal control,model, medication and EA groups, with 15 rats in each group. Rat model of AGA was established by injection of 0.2 mL sodium urate crystal suspension into the left knee joint cavity. The rats in the medication group were treated with colchicine by gavage(0.3 mg·kg-1·d-1), and the rats in the EA group were treated with EA at the left "Sanyinjiao" (SP6) and "Zusanli"(ST36) for 10 min each time, once a day for a week. The Coderre gait grading standard was used to score the gait of rats. The pathological morphology of synovial tissue of the left knee joint was observed by H.E. staining. The expression levels of Cathepsin-B protein and Nod-like receptor pyrin domain 3(NLRP3), apoptosis-associated speck-like protein containing CARD (ASC),Caspase-1, interleukin-1ß(IL-1ß) and IL-18 mRNAs were detected by Western blot and real-time fluorescence quantitative PCR, respectively. RESULTS: Compared with the normal control group, the degree of synovitis infiltration in the model group was more serious. And the gait score，the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3,ASC,Caspase-1, IL-1ß,IL-18 were significantly increased (P<0.01).After the interventions, the degree of inflammatory infiltration was mild, The gait score, the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3 and ASC,Caspase-1，IL-1ß,IL-18 were significantly decreased in both medication and EA groups in contrast to the model group (P<0.01, P<0.05). Compared with medication group, the mRNA expression levels of Caspase-1 and IL-18 in the EA group were increased (P<0.05). CONCLUSION: EA may inhibit the activation of NLRP3 inflammasome by reducing the activity of Cathepsin-B in the synovium of the knee joint, so as to treat AGA.

IL-6 promotes collagen-induced arthritis by activating the NLRP3 inflammasome through the cathepsin B/S100A9-mediated pathway.

Rheumatoid arthritis (RA) is an inflammatory disease with symmetric polyarthritis. IL-6 and NLRP3 inflammasome in macrophages contribute to the pathogenesis of RA. This study aimed to investigate the relationship between IL-6 and the NLRP3 inflammasome in RA. Here, we found that IL-6 inhibition reduced NLRP3 inflammasome activation in mice with collage-induced arthritis (CIA). In vitro studies showed that IL-6 directly induced NLRP3 inflammasome activation via cathepsin B (CTSB) in the presence of ATP. In addition, S100A9 induced by ATP stimulation promoted the interaction of CTSB and NLRP3 to activate the NLRP3 inflammasome. Our findings show a novel mechanism of NLRP3 inflammasome activation by IL-6 that may lead to a potential therapy for RA by interrupting the interaction between IL-6 and the NLRP3 inflammasome.

Mechanism of action of Panax notoginoside against lung cancer in mice based on response to CTSB gene.

BACKGROUND: This study aimed to investigate the mechanism of action of Panax notoginoside (PNS) against lung cancer and inhibition of lung cancer cell proliferation by the drug at different concentrations in a mouse model, considering the cathepsin B (CTSB) gene as a target. METHODS: The mice were randomly assigned into the following five groups: normal control, tumor-bearing, low-dose Panax notoginoside (TSPN), medium-dose TSPN, and high-dose TSPN. All mice were treated with physiological saline or TSPN at different concentrations for 28 days consecutively by gavage. The tumor size was measured, the tumor growth was observed, and the survival curve was drawn. At different time points, the expression of the CTSB gene was detected using quantitative fluorescent polymerase chain reaction, Western blot analysis, and indirect immunofluorescence. The serum indices, such as carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and Soluble fragment of cytokeratin 19 (CYFRA21), were detected by enzyme-linked immunosorbent assay. RESULTS: In vivo, PNS could directly inhibit the expression of the CTSB gene in tumors of mice, limit tumor growth, and alter tumor-related indices, such as CEA, NSE, and CYFRA21 levels, in the serum to different extents simultaneously. CONCLUSION: CTSB gene was closely related to the pathogenesis of lung cancer. PNS could act on the CTSB gene, downregulate the expression of CTSB in lung cancer cells, inhibit the proliferation and invasion of tumors, and prolong the survival period.

Isoimperatorin (ISO) reduces melanin content in keratinocytes via miR-3619/CSTB and miR-3619/CSTD axes.

Melanin metabolism disorders may cause severe impacts on the psychological and social activities of patients. Different from the other two steps of melanin metabolism, namely synthesis and transport, little has been known about the mechanism of melanin degradation. Isoimperatorin (ISO) suppressed the activity of tyrosinase, an essential enzyme in melanin biosynthesis, hence, we investigated the effects and mechanism of ISO in melanin reduction. ISO stimulation significantly reduces the melanin contents and PMEL 17 protein levels; meanwhile, the activity and the protein levels of two critical lysosomal enzymes, Cathepsin B and Cathepsin D, can be significantly increased by ISO treatment. MiR-3619 inhibited the expression of CSTB and CSTD, therefore affecting ISO-induced degradation of melanin. In summary, ISO reduces the melanin content via miR-3619/CSTB and miR-3619/CSTD axes. ISO could be a potent skin-whitening agent, which needs further in vivo and clinical investigation.

Real-time in situ monitoring of signal molecules' evolution in apoptotic pathway via Au-Se bond constructed nanoprobe.

In cell signal transduction pathways, a series of biochemical reactions and interactions between proteins guarantee physiological responses indicating cell functionality. However, there are a variety of upstream and downstream signal molecules in these pathways with multiple levels of cross-regulation, making it difficult to sequential visualize their relationships in living organisms in complex environments. To investigate the interrelationships among intracellular signaling pathways, a Au-Se bonded nanoprobe with extraordinary stability and strong anti-interference ability was designed and prepared to monitor the evolution of two kinds of apoptosis biomarkers in real time. Two different peptide chains decorated with two dyes were functionalized on the surface of Au nanoparticles (NPs) via Au-Se bonds. These peptide chains can be respectively cleaved by upstream cathepsin B proteins and downstream caspase-3 proteins to trigger fluorescence recovery. Moreover, when the living cells were stimulated to induce the apoptotic pathway, cathepsin B and caspase-3 were activated in turn with signals sequentially recovered at 2 and 4 h, respectively. This fluorescent nanoprobe can be used in complicated biological systems to achieve real-time in situ monitoring of the sequential activation of signal molecules in intracellular pathways and provides a novel approach for the future investigation of protein interactions in vivo.

Folate deficiency-induced oxidative stress contributes to neuropathy in young and aged zebrafish--implication in neural tube defects and Alzheimer's diseases.

Folate is a nutrient essential for the development, function and regeneration of nervous systems. Folate deficiency has been linked to many neurological disorders including neural tube defects in fetus and Alzheimer's diseases in the elderly. However, the etiology underlying these folate deficiency-associated diseases is not completely understood. In this study, zebrafish transgenic lines with timing and duration-controllable folate deficiency were developed by ectopically overexpressing a recombinant EGFP-γ-glutamyl hydrolase (γGH). Impeded neural crest cell migration was observed in the transgenic embryos when folate deficiency was induced in early stages, leading to defective neural tube closure and hematopoiesis. Adding reduced folate or N-acetylcysteine reversed the phenotypic anomalies, supporting the causal link between the increased oxidative stress and the folate deficiency-induced abnormalities. When folate deficiency was induced in aged fish accumulation of beta-amyloid and phosphorylated Tau protein were found in the fish brain cryo-sections. Increased autophagy and accumulation of acidic autolysosome were apparent in folate deficient neuroblastoma cells, which were reversed by reduced folate or N-acetylcysteine supplementation. Decreased expression of cathepsin B, a lysosomal protease, was also observed in cells and tissue with folate deficiency. We concluded that folate deficiency-induced oxidative stress contributed to the folate deficiency-associated neuropathogenesis in both early and late stages of life.

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus).

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.

Qushi Huayu Decoction (祛湿化瘀方) inhibits protein and gene expression of cathepsin B in HepG2 cells induced by free fatty acids.

OBJECTIVE: To study the experimental efficacy of Qushi Huayu Decoction (祛湿化瘀方,QHD) on, protein and gene expression of cathepsin B (ctsb) in HepG2 cells induced by free fatty acids (FFAs). METHODS: The model of HepG2 steatosis and tumor necrosis factor-α (TNF-α) secretion was induced by long-chain FFAs. HepG2 cells were divided into 4 groups: control group (group C), model group (group M), low-dose QHD group (group L) and high-dose QHD group (group H). Long-chain FFAs were added to groups M, L and H. The 10% blank-control serum was added to group C and M, while 5% and 10% QHD-containing sera were added to group L and H, respectively. The levels of serum TNF-α and cellular triglyceride (TG) were detected. Cellular p-IκB and ctsb expression were detected using Western blot and PCR. The expression and distribution of ctsb were observed by immunofluorescence. RESULTS: After incubating with FFA for 24 h, TG deposition in HepG2, TNF-α content in cell supernatant, the protein expression of cellular ctsb and P-IκB, as well as mRNA expression of ctsb increased markedly in group M compared with group C (P<0.05, P<0.01). Compared with group M, TG deposition, the expression of cellular ctsb, P-IκB and ctsb mRNA in groups L and H, as well as TNF-α content in group H, decreased significantly (P<0.05). Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA. In this study, these above-mentioned changes were inhibited markedly in groups L and H. CONCLUSION: QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.

Influence of glutamine infusion on ubiquitin, caspase-3, cathepsins L and B, and m-calpain expression in sheep with nutritionally induced metabolic acidosis.

Provision of AA has shown success in attenuating proteolytic activity in monogastrics suffering from metabolic acidosis. However, it is unknown whether AA supplementation can provide any beneficial effects to ruminants with nutritionally induced metabolic acidosis. The objective of the current study was to examine the effects of glutamine infusion on various protein degradation components across several tissues in sheep with induced metabolic acidosis. Sheep were assigned to a randomized complete block design with 2 x 2 factorial arrangement of treatments (n = 6 sheep/treatment) consisting of a control or acidosis diet, and receiving a saline or L-glutamine infusion. Sheep were fed diets for 10 d and slaughtered on d 11. Liver, kidney, and muscle samples were collected at slaughter and examined for relative messenger RNA (mRNA) expression of ubiquitin, C8, E2, cathepsin L, cathepsin B, caspase-3, and m-calpain, as well as protein expression of ubiquitin. Relative mRNA expression of C8 (P = 0.02), E2 (P = 0.06), and ubiquitin (P = 0.07) was less in kidney in acidotic vs. control sheep. Additionally, mRNA expression of m-calpain in kidney was greater (P = 0.01) as a result of glutamine infusion. There were no significant alterations (P > 0.10) in mRNA of any component as a result of acidosis in the liver or muscle. This study demonstrates the inability of metabolic acidosis to increase expression of the ubiquitin-mediated proteolytic pathway in skeletal muscle; however, downregulation of renal mRNA expression of these components is apparent during the induction of metabolic acidosis.

[Effects of Qushi Huayu Decoction on cathepsin B and tumor necrosis factor-alpha expression in rats with non-alcoholic steatohepatitis].

OBJECTIVE: To study the mechanism of Qushi Huayu Decoction (QHD), a compound of traditional Chinese herbal medicine, in prevention and treatment of non-alcoholic steatohepatitis (NASH). METHODS: Thirty-five Wistar male rats were randomly divided into normal group, untreated group, QHD group and Ganle (diisopropylamine dichloroacetate) group. The rats except those in normal group were subcutaneously injected with carbon tetrachloride (CCl(4)) for 4 weeks (twice per week) and simultaneously fed with high-fat and low-protein diet for 2 weeks to induce NASH. Then, the rats were administrated with QHD, Ganle, or distilled water for 2 weeks, respectively. After harvest, alanine aminotransferase (ALT) activity and tumor necrosis factor-alpha (TNF-alpha) content in serum as well as triglyceride (TG) and free fatty acid (FFA) in liver tissue were evaluated, and relativity analysis among these parameters was performed. Cathepsin B (Ctsb), phospho-inhibitor kappa B (P-IkappaB), TNF-alpha protein expressions in liver tissue were assayed with western-blot. The expression and distribution of ctsb in liver tissue were observed with immunohistochemical method. RESULTS: The contents of TG, FFA and activity of ALT were significantly decreased in QHD group. While in the Ganle group, only the activity of ALT in serum was decreased significantly. Expressions of Ctsb, P-IkappaB and TNF-alpha proteins in liver tissues and serum TNF-alpha level were all enhanced in untreated group which, however, were significantly inhibited in the QHD group. And as expected, there were significant relativities among contents of TG in liver tissues and the content of FFA in liver tissue and activity of ALT in serum, content of TNF-alpha in serum and content of FFA in liver tissue and activity of ALT in serum. CONCLUSION: The inhibiting effects of QHD on fat deposition and inflammation in liver are related with its inhibition on the "FFA-Ctsb-TNF-alpha" pathway of lipo-toxicity.

Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein.

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.

Effects of serum deprivation on expression of proteolytic-related genes in chick myotube cultures.

We previously reported that serum deprivation stimulates myofibrillar proteolysis in chick myotubes. In the present study, we examined the effect of serum deprivation on expression of the proteolytic-related genes (ubiquitin, proteasome, calpains, and cathepsin B) by real-time PCR of cDNA in chick myotubes. Myotubes were incubated with serum-free medium for 24 h. Ubiquitin and proteasome subunits (C1 and C2) and calpains (m-, mu-, and p94/calpain-3) but not cathepsin B mRNA expression were increased by serum deprivation. These results indicate that serum deprivation stimulates ubiquitin-proteasome and calpain proteolytic pathways, resulting in an increase in myofibrillar proteolysis in chick myotubes.

Cathepsin B and L are involved in degradation of prions in GT1-1 neuronal cells.

In scrapie-infected cells, the abnormal isoform of the prion protein, PrP(Sc), accumulates in endosomes/lysosomes. In this study, the involvement of two lysosomal proteases, cathepsin B and L, in cellular processing of PrP(Sc) was analyzed in immortalized neuronal gonadotropin-releasing hormone cells (GT1-1) infected with scrapie. Treatment with inhibitors of either cathepsin B or L resulted in accumulation of PrP(Sc). Such an increased accumulation also occurred when the activities of both cathepsins were inhibited using RNA interference. We conclude that cathepsin B and L are involved in the degradation of PrP(Sc) in scrapie-infected GT1-1 cells and that they can compensate for each other's functions. This study shows that specific proteases, abundantly present in neurons, have the capacity to degrade PrP(Sc).

Molecular regulation of human cathepsin B: implication in pathologies.

Cathepsin B is a papain-family cysteine protease that is normally located in lysosomes, where it is involved in the turnover of proteins and plays various roles in maintaining the normal metabolism of cells. This protease has been implicated in pathological conditions, e.g., tumor progression and arthritis. In disease conditions, increases in the expression of cathepsin B occur at both the gene and protein levels. At the gene level, the altered expression results from gene amplification, elevated transcription, use of alternative promoters and alternative splicing. These molecular changes lead to increased cathepsin B protein levels and in turn redistribution, secretion and increased activity. Here we focus on the molecular regulation of cathepsin B and attendant implications for tumor progression and arthritis. The potential of cathepsin B as a therapeutic target is also discussed.

Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases.

Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.

Molecular characterization of three gut genes from Glossina morsitans morsitans: cathepsin B, zinc-metalloprotease and zinc-carboxypeptidase.

Insect gut enzymes are involved in digestion of dietary proteins. Additionally, these enzymes have been implicated in the process of pathogen establishment in several insects including the tsetse fly (Diptera:Glossinidae), which is the vector for African trypanosomes. Both the male and female tsetse can transmit trypanosomes and are strict blood feeders during all stages of their development. Here, we describe the molecular characterization of three gut genes: cathepsin B (GmCatB), zinc-metalloprotease (GmZmp) and zinc-carboxypeptidase (GmZcp). The cDNA for GmCatB encodes a protein for 340 amino acids with a predicted molecular mass of 38.2 kDa, while the 854 bp GmZmp cDNA encodes a protein of 254 amino acids with a molecular mass of 29 kDa. The GmZcp cDNA is 1319 bp in length and has a 354 amino acids open reading frame for coding a 40 kDa protein. All three cDNAs have signal peptide sequences associated with their N-terminal domains and structure analysis indicates that GmCatB and GmZmp are expressed as zymogens with pro-domains proteolytically removed for activity. The activation domain associated with the carboxypeptidase sequences is lacking in GmZcp. While GmCatB transcription is constitutive, teneral flies express very low levels of transcripts for GmZmp and GmZcp prior to the first bloodmeal. Transcription of all genes is induced and remains high throughout the digestion cycle within a few hours following the first bloodmeal ingestion. Both GmCatB and GmZcp are parasite responsive, with the expression of both genes being higher in trypanosome infected flies.

Cloning of cDNA for cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), and characterization of recombinant CBBP.

Previously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.