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Sample records for multiple nucleopolyhedrovirus cathepsin

  1. Interactions between Meteorus pulchricornis and Spodoptera exigua multiple nucleopolyhedrovirus.

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    Guo, Hui-Fang; Fang, Ji-Chao; Zhong, Wan-Fang; Liu, Bao-Sheng

    2013-01-01

    Baculoviruses may interact with parasitoids in the same host. A previous study has shown that infection of larvae with Spodoptera litura nucleopolyhedrovirus (SpltNPV) was deleterious to the survival and development of Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae). In this paper, the interactions between M. pulchricornis and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), a permissive host of the virus and parasitoid, were investigated. The results showed that the effect of M. pulchricornis on SeMNPV and the effect of the virus on the parasitoid both depended on the concentration of the virus and the interval between viral infection and parasitism. Whether S. exigua was treated with the parasitoid and virus simultaneously or 1 day apart, the biological activities of 10(5), 10(6), and 10(7) OBs/mL SeMNPV were all significantly improved by M. pulchricornis. In contrast, the biological activity of 10(3) OBs/mL SeMNPV was significantly decreased when the host was exposed to the virus and parasitoid simultaneously. Regarding the impact of SeMNPV on M. pulchricornis, exposing the host to the parasitoid and SeMNPV with concentrations lower than 10(6) occlusion bodies (OBs)/mL produced no negative effects on the parasitoid. The results also showed that ingestion of SeMNPV by adult stage M. pulchricornis significantly increased the number of parasitoid offspring that successfully emerged from the host. Furthermore, M. pulchricornis was found to transmit SeMNPV among populations of S. exigua. Taken together, these findings indicate that M. pulchricornis integrated with an appropriate concentration of SeMNPV has the potential to improve the efficacy of biological control against S. exigua.

  2. Host-range expansion of Spodoptera exigua multiple nucleopolyhedrovirus to Agrotis segetum larvae when the midgut is bypassed

    NARCIS (Netherlands)

    Jakubowska, A.K.; Lynn, D.E.; Herrero, S.; Vlak, J.M.; Oers, van M.M.

    2010-01-01

    Given the high similarity in genome content and organization between Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum nucleopolyhedrovirus (AgseNPV), as well as the high percentages of similarity found between their 30 core genes, the specificity of these NPVs was

  3. A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations

    Science.gov (United States)

    V.V. Martemyanov; J.D. Podgwaite; I.A. Belousova; S.V. Pavlushin; J.M. Slavicek; O.A. Baturina; M.R. Kabilov; A.V. Ilyinykh

    2017-01-01

    The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute...

  4. ac18 is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus

    International Nuclear Information System (INIS)

    Wang Yanjie; Wu Wenbi; Li Zhaofei; Yuan Meijin; Feng Guozhong; Yu Qian; Yang Kai; Pang Yi

    2007-01-01

    orf18 (ac18) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, an ac18 knockout AcMNPV bacmid was generated to determine the role of ac18 in baculovirus life cycle. After transfection of Sf-9 cells, the ac18-null mutant showed similar infection pattern to the parent virus and the ac18 repair virus with respect to the production of infectious budded virus, occlusion bodies, or the formation of nucleocapsids as visualized by electron microscopy. The deletion mutant did not reduce AcMNPV infectivity for Trichoplusia ni in LD 50 bioassay; however, it did take 24 h longer for deleted mutant to kill T. ni larvae than wild-type virus in LT 50 bioassay. Our results demonstrate that ac18 is not essential for viral propagation both in vitro and in vivo, but it may play a role in efficient virus infection in T. ni larvae

  5. Comparative proteomics analysis of Spodoptera frugiperda cells during Autographa californica multiple nucleopolyhedrovirus infection.

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    Yu, Qian; Xiong, Youhua; Gao, Hang; Liu, Jianliang; Chen, Zhiqiang; Wang, Qin; Wen, Dongling

    2015-08-04

    Increasing evidence sugggest that in addition of balculovirus controling insect host, host cells also responds to balculovirus infection. However, compared to existing knowledge on virus gene, host cell responses are relatively poorly understood. In this study, Spodoptera frugiperda (Sf9) cells were infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The protein composition and protein changes of Spodoptera frugiperda (Sf9) cells of different infection stages were analysed by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 Sf9 proteins were identified by iTRAQ and 413 proteins were found as more than 1.5-fold changes in abundance. The 413 proteins were categorised according to GO classification for insects and were categorised into: biological process, molecular function and cellular component. The determination of the protein changes in infected Sf9 cells would help to better understanding of host cell responses and facilitate better design of this virus-host cell interaction in pest insect control and other related fields.

  6. Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

    International Nuclear Information System (INIS)

    Liu Chao; Li Zhaofei; Wu Wenbi; Li Lingling; Yuan Meijin; Pan Lijing; Yang Kai; Pang Yi

    2008-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc ac53KO-PH-GFP ) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc ac53KO-PH-GFP could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are present in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production

  7. A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

    Science.gov (United States)

    Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

    2017-08-01

    In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

  8. A Comparison of Infectivity between Polyhedra of the Spodoptera litura Multiple Nucleopolyhedrovirus Before and After Passage Through the Gut of the Stink Bug, Eocanthecona furcellata

    OpenAIRE

    Gupta, R. K.; Gani, Mudasir; Jasrotia, P.; Srivastava, K.; Kaul, V.

    2014-01-01

    Infectivity of polyhedra of Spodoptera litura multiple nucleopolyhedrovirus before and after passage through the gut of the predatory stink bug, Eocanthecona furcellata Wolff (Hemiptera: Pentatomidae) was compared through field bioassay studies. Three sets of E. furcellata were used for bioassays and these were allowed to feed on a single meal of five third instar Oriental leaf worm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), that were infected with polyhedra before passage, afte...

  9. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Qianlong [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Blissard, Gary W. [Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, United State (United States); Liu, Tong-Xian [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Zhaofei, E-mail: zhaofeili73@outlook.com [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China)

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  10. Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection

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    Wenqiang Wei

    2016-05-01

    Full Text Available At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

  11. Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV

    NARCIS (Netherlands)

    Jakubowska, A.K.; Peters, S.A.; Ziemnicka, J.; Vlak, J.M.; Oers, van M.M.

    2006-01-01

    The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147 544 bp and has a G+C content of 45¿7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins

  12. Geographic isolates of Lymantria dispar multiple nucleopolyhedrovirus: Genome sequence analysis and pathogenicity against European and Asian gypsy moth strains.

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    Harrison, Robert L; Rowley, Daniel L; Keena, Melody A

    2016-06-01

    Isolates of the baculovirus species Lymantria dispar multiple nucleopolyhedrovirus have been formulated and applied to suppress outbreaks of the gypsy moth, L. dispar. To evaluate the genetic diversity in this species at the genomic level, the genomes of three isolates from Massachusetts, USA (LdMNPV-Ab-a624), Spain (LdMNPV-3054), and Japan (LdMNPV-3041) were sequenced and compared with four previously determined LdMNPV genome sequences. The LdMNPV genome sequences were collinear and contained the same homologous repeats (hrs) and clusters of baculovirus repeat orf (bro) gene family members in the same relative positions in their genomes, although sequence identities in these regions were low. Of 146 non-bro ORFs annotated in the genome of the representative isolate LdMNPV 5-6, 135 ORFs were found in every other LdMNPV genome, including the 37 core genes of Baculoviridae and other genes conserved in genus Alphabaculovirus. Phylogenetic inference with an alignment of the core gene nucleotide sequences grouped isolates 3041 (Japan) and 2161 (Korea) separately from a cluster containing isolates from Europe, North America, and Russia. To examine phenotypic diversity, bioassays were carried out with a selection of isolates against neonate larvae from three European gypsy moth (Lymantria dispar dispar) and three Asian gypsy moth (Lymantria dispar asiatica and Lymantria dispar japonica) colonies. LdMNPV isolates 2161 (Korea), 3029 (Russia), and 3041 (Japan) exhibited a greater degree of pathogenicity against all L. dispar strains than LdMNPV from a sample of Gypchek. This study provides additional information on the genetic diversity of LdMNPV isolates and their activity against the Asian gypsy moth, a potential invasive pest of North American trees and forests. Published by Elsevier Inc.

  13. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

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    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome.

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    Ren, He-Lin; Hu, Yuan; Guo, Ya-Jun; Li, Lu-Lin

    2016-06-01

    Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus (PlxyGV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, and compared with homologous late gene promoters of AcMNPV in Sf9 cells. In transient expression assays, all PlxyGV late promoters were activated in cells transfected with the individual reporter plasmids together with an AcMNPV bacmid. In infected cells, reporter gene expression levels with the promoters of PlxyGV e18 and AcMNPV vp39 and gp41 were significantly higher than those of the corresponding AcMNPV or PlxyGV promoters, which had fewer late promoter motifs. Observed expression levels were lower for the PlxyGV p6.9, pk1, gran, p10a, and p10b promoters than for the corresponding AcMNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the AcMNPV polh, p10, and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of PlxyGV gran, p10c, and pk1. The results of this study demonstrated that PlxyGV late gene promoters could be effectively activated by the RNA polymerase from AcMNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.

  15. Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus

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    Santiago Haase

    2015-03-01

    Full Text Available Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh was replaced by a bacterial β-galactosidase (lacZ gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+ AgMNPV expressing the green fluorescent protein gene (gfp. This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties.

  16. The "11K" gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus.

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    Beperet, Inés; Simón, Oihane; Williams, Trevor; López-Ferber, Miguel; Caballero, Primitivo

    2015-05-01

    The "11K" gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and sf95) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair bacmids were generated from a bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were ∼15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and sf95 resulted in a slower speed-of-kill by 9h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation

    International Nuclear Information System (INIS)

    Ke Jianhao; Wang Jinwen; Deng Riqiang; Wang Xunzhang

    2008-01-01

    Although orf66 (ac66) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in all sequenced lepidopteran baculovirus genomes, its function is not known. This paper describes generation of an ac66 knockout AcMNPV bacmid mutant and analyses of the influence of ac66 deletion on the virus replication in Sf-9 cells so as to determine the role of ac66 in the viral life cycle. Results indicated that budded virus (BV) yields were reduced over 99% in ac66-null mutant infected cells in comparison to that in wild-type virus infected cells. Optical microscopy revealed that occlusion body synthesis was significantly reduced in the ac66 knockout bacmid-transfected cells. In addition, ac66 deletion interrupted preoccluded virion synthesis. The mutant phenotype was rescued by an ac66 repair bacmid. On the other hand, real-time PCR analysis indicated that ac66 deletion did not affect the levels of viral DNA replication. Electron microscopy revealed that ac66 is not essential for nucleocapsid assembly, but for the efficient transport of nucleocapsids from the nucleus to the cytoplasm. These results suggested that ac66 plays an important role for the efficient exit of nucleocapsids from the nucleus to the cytoplasm for BV synthesis as well as for preoccluded virion and occlusion synthesis

  18. A comparison of infectivity between polyhedra of the Spodoptera litura multiple nucleopolyhedrovirus before and after passage through the gut of the stink bug, Eocanthecona furcellata.

    Science.gov (United States)

    Gupta, R K; Gani, Mudasir; Jasrotia, P; Srivastava, K; Kaul, V

    2014-01-01

    Infectivity of polyhedra of Spodoptera litura multiple nucleopolyhedrovirus before and after passage through the gut of the predatory stink bug, Eocanthecona furcellata Wolff (Hemiptera: Pentatomidae) was compared through field bioassay studies. Three sets of E. furcellata were used for bioassays and these were allowed to feed on a single meal of five third instar Oriental leaf worm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), that were infected with polyhedra before passage, after passage, and healthy (control) larvae 1 day prior to the trial. The predators were subsequently released on cabbage plants that were infested with 100 healthy S. litura larvae. The median lethal dose (LD50) and survival time (ST50) values before and after passage through the gut were not significantly different. Additional mortality due to virus infection increased 13- 17% before and after treatments but within these treatments the mortality did not vary significantly. It was concluded that E. furcellata disseminated the virus through their feces into the ecosystem and infectivity of the SpltMNPV was not altered after passage through the gut of the predator. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

  19. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    International Nuclear Information System (INIS)

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-01-01

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  20. Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies▿

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    Simón, Oihane; Williams, Trevor; Asensio, Aaron C.; Ros, Sarhay; Gaya, Andrea; Caballero, Primitivo; Possee, Robert D.

    2008-01-01

    The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB. PMID:18550678

  1. Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles.

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    Ya-Jun Guo

    Full Text Available In this study, Autographa californica multiple nucleopolyhedrovirus ac75 was functionally characterized. Ac75 has homologs in all sequenced genomes of alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. It was determined to encode a protein that is associated with the nucleocapsid of budded virus and with both envelope and nucleocapsids of occlusion-derived virus. Sf9 cells transfected by an ac75-knockout bacmid resulted in the infection being restricted to single cells. No budded virus were detected although viral DNA replication and late gene expression were unaffected. Electron microscopy revealed that the virogenic stroma, nucleocapsids and occlusion bodies appeared normal in the cells transfected by an ac75-knockout bacmid. However, the nucleocapsids were unenveloped, the occlusion bodies did not contain any virions or nucleocapsids, and no nucleocapsids were found outside the nucleus or spanning the nuclear membrane. In addition, de novo intranuclear membrane microvesicles that are the precursor of occlusion-derived virus envelopes were absent in the nuclei of transfected cells. Confocal microscopy showed that AC75 protein appeared in the cytoplasm as early as 6 hours post infection. It localized to the ring zone at the periphery of the nucleus from 15 to 24 hours post infection and demonstrated light blocky cloud-like distribution in the center of the nucleus. AC75 was found to co-immunoprecipitate with BV and ODV associated envelope protein ODV-E25. The data from this study suggest that ac75 is essential for induction of the intranuclear membrane microvesicles, it appears to be required for the intranuclear envelopment of nucleocapsids, and is also essential for egress of nucleocapsids from the nuclei, in infected cells.

  2. Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi.

    Science.gov (United States)

    Sun, Yu-Xuan; Zhu, Bao-Jian; Tang, Lin; Sun, Yu; Chen, Chen; Nadeem Abbas, Muhammad; Wang, Lei; Qian, Cen; Wei, Guo-Qing; Liu, Chao-Liang

    2017-11-01

    Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of A. pernyi. Copyright © 2017. Published by Elsevier Inc.

  3. The sf32 unique gene of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV is a non-essential gene that could be involved in nucleocapsid organization in occlusion-derived virions.

    Directory of Open Access Journals (Sweden)

    Inés Beperet

    Full Text Available A recombinant virus lacking the sf32 gene (Sf32null, unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV, was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac. Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration, speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs.

  4. Cathepsin D inhibitors

    Directory of Open Access Journals (Sweden)

    M. Gacko

    2007-11-01

    Full Text Available Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirectproduct, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes withcathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme.Methods used to determine the activity and concentration of these inhibitors and their analytical, preparative and therapeuticapplications are discussed.

  5. Characterization of Bombyx mori nucleopolyhedrovirus Bm17.

    Science.gov (United States)

    Shen, Hongxing; Wang, Rudu; Han, Qinggong; Zhang, Wen; Nin, Bin; Zhou, Yang; Shao, Shihe; Yao, Qin; Chen, Keping; Liu, Xiaoyong

    2013-10-01

    Open reading frame17 (Bm17) of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this report, we describe the characterization of Bm17. Reversed transcriptive-PCR (RT-PCR) and Western blot analysis demonstrated that Bm17 was expressed as a late gen. Immunofluorescence analysis by confocal microscopy showed that BM17 protein was localized on cytoplasm and nucleus of infected cells. These results show that BM17 was a late protein localized in cytoplasm and nucleus. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Role of Cathepsin S in Periodontal Inflammation and Infection

    Directory of Open Access Journals (Sweden)

    S. Memmert

    2017-01-01

    Full Text Available Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

  7. Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

    Science.gov (United States)

    Cheriyath, V; Kuhns, M A; Kalaycio, M E; Borden, E C

    2011-03-15

    Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas. At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (Pmediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

  8. Spodoptera exigua nucleopolyhedrovirus is not infectious for Agrotis segetum larvae per os, but only after intrahemocoelic injection

    NARCIS (Netherlands)

    Jakubowska, A.K.; Ince, I.A.; Herrero, S.; Vlak, J.M.; Oers, van M.M.

    2009-01-01

    Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum NPV are close relatives, but distinct baculovirus species. Their genomic organization is remarkably similar. The cross infectivity of these two viruses for S. exigua and A. segetum larvae has been analyzed. AgseNPV was able

  9. Cathepsin L in human meningiomas

    International Nuclear Information System (INIS)

    Trinkaus, M.; Lah, T.T.; Vranic, A.; Dolenc, V.V.

    2003-01-01

    Background. Although meningiomas are considered as benign tumours, about 10% comprise a subgroup of a typical meningiomas, classified as WHO grade II, with greater likelihood of recurrences and/or aggressive behaviour, including the possibility of brain tissue invasion. The lysosomal cysteine endopeptidase cathepsin L plays a role in tumour cell invasion and malignant progression of cancer, and has been suggested as a prognostic marker for certain types of tumours. Results. In our study, we compared the expression of cathepsin L in 30 meningiomas with their clinical invasiveness. Cathepsin L was determined by immunohistochemical analysis, quantitative real-time RT-PCR and Northern blot. We showed that expression of cathepsin L protein was significantly higher (p=0.019) in 9 atypical than in 21 benign meningiomas. Within the group of benign meningiomas, expression of cathepsin L was significantly lower in the transitional histological subtype. We measured the levels of cathepsin LA type of RNA splicing variants: LA, LAI and LAII, but not LAIII and not the LB variant, the latter being several times lower than the LA type. In contrast to protein levels, the levels of cathepsin LA, AI, AII RNA variants did not differ between histological subtypes or between benign and a typical meningiomas. The expression of total measured cathepsin LA, AI, AII RNA variants in the samples, taken from the centre and the periphery of the tumours, also showed no statistically significant differences. Conclusions. These results indicate that cathepsin L protein over-expression may contribute to the development of the aggressive and possibly invasive character of a typical meningiomas and that it may be up regulated at the translational level. (author)

  10. Transmission of Different Nucleopolyhedroviruses by Two Ectoparasitoids – Bracon hebetor Say (Hymenoptera: Braconidae) and Euplectrus plathypenae (Howard) (Hymenoptera: Eulophidae)

    OpenAIRE

    Emanouela E. Stoianova; Nikolay A. Balevski

    2010-01-01

    The transmission of nucleopolyhedroviruses (NPVs) of Autographa gamma (AgNPV), Mamestra brassicae (MbNPV), Lacanobia oleraceae (LoNPV), Helicoverpa armigera (HaNPV) and Xantia c-nigrum (XnNPV) to their relevant larvae by the ectoparasitoid Bracon hebetor and the transmission of the multiple-enveloped NPVs of Spodoptera exigua (SeMNPV) and Spodoptera frugiperda (SfMNPV) by the ectoparasitoid Euplectrus plathypenae was examined. Two methods of contamination of the both parasitoids (exposure to ...

  11. Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno.

    Science.gov (United States)

    Rodríguez, Vanina Andrea; Belaich, Mariano Nicolás; Gómez, Diego Luis Mengual; Sciocco-Cap, Alicia; Ghiringhelli, Pablo Daniel

    2011-02-01

    Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine.

    Science.gov (United States)

    Szymczak, Mariusz

    2016-08-01

    This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency-high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology. © 2016 Institute of Food Technologists®

  13. Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

    Science.gov (United States)

    Platt, Manu O

    2017-01-01

    Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

  14. Genomic diversity of Bombyx mori nucleopolyhedrovirus strains.

    Science.gov (United States)

    Xu, Yi-Peng; Cheng, Ruo-Lin; Xi, Yu; Zhang, Chuan-Xi

    2013-07-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects the domestic silkworm. In this study, six BmNPV strains were compared at the whole genome level. We found that the number of bro genes and the composition of the homologous regions (hrs) are the two primary areas of divergence within these genomes. When we compared the ORFs of these BmNPV variants, we noticed a high degree of sequence divergence in the ORFs that are not baculovirus core genes. This result is consistent with the results derived from phylogenetic trees and evolutionary pressure analyses of these ORFs, indicating that ORFs that are not core genes likely play important roles in the evolution of BmNPV strains. The evolutionary relationships of these BmNPV strains might be explained by their geographic origins or those of their hosts. In addition, the total number of hr palindromes seems to affect viral DNA replication in Bm5 cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex.

    Science.gov (United States)

    Dong, Zhan-Qi; Zhang, Jun; Chen, Xue-Mei; He, Qian; Cao, Ming-Ya; Wang, La; Li, Hai-Qing; Xiao, Wen-Fu; Pan, Cai-Xia; Lu, Cheng; Pan, Min-Hui

    2014-05-12

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF79 (Bm79) encodes an occlusion-derived virus (ODV)-specific envelope protein, which is a homologue of the per os infectivity factor 4 (PIF4) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To investigate the role of ORF79 in the BmNPV life cycle, a Bm79 knockout virus (vBm(Bm79KO)) was constructed through homologous recombination in Escherichia coli. Viral DNA replication, budded virus (BV) production and polyhedra formation were unaffected by the absence of BM79. However, results of the larval bioassay demonstrated that the Bm79 deletion resulted in a complete loss of per os infection. Immunofluorescence analysis showed that BM79 localized at the innernuclear membrane of infected cells through its N-terminal sorting motif (SM). Further bimolecular fluorescence protein complementation and co-immunoprecipitation assays demonstrated the interaction of BM79 with PIF1, PIF2, PIF3 and ODV-E66. Thus, BM79 plays an important role in per os infection and is associated with the viral PIF complex of BmNPV. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

    Science.gov (United States)

    Han, Yingying; Xia, Hengchuan; Tang, Qi; Lü, Peng; Ma, Shangshang; Yang, Yanhua; Shao, Dandan; Ma, Quanbing; Chen, Keping

    2014-07-01

    The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

  17. Molecular characterization of Agrotis segetum nucleopolyhedrovirus from Poland

    NARCIS (Netherlands)

    Jakubowska, A.K.; Oers, van M.M.; Ziemnicka, J.; Lipa, J.J.; Vlak, J.M.

    2005-01-01

    The turnip moth, Agrotis segetum (Lepidoptera, Noctuidae), is an important pest insect in Europe, Asia, and Africa. We have genetically characterized and classified a nucleopolyhedrovirus isolated from A. segetum larvae in Poland (AgseNPV-P). The restriction pattern of AgseNPV-P was distinct from an

  18. The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome

    NARCIS (Netherlands)

    Chen, X.; IJkel, W.F.J.; Tarchini, R.; Sun, X.; Sandbrink, H.; Wang, H.; Peters, S.; Zuidema, D.; Klein Lankhorst, R.; Vlak, J.M.; Hu, Z.

    2001-01-01

    The nucleotide sequence of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) DNA genome was determined and analysed. The circular genome encompasses 131 403 bp, has a G C content of 39.1 molnd contains five homologous regions with a unique pattern of repeats.

  19. [Research progress on cathepsin F of parasitic helminths].

    Science.gov (United States)

    Qu, Zi-Gang; Fu, Bao-Quan

    2013-10-01

    Cathepsin F is an important member of papain-like subfamily in cysteine protease family. Cathepsin F of helminth parasites can hydrolyze the specific substrate, degrade host protein such as hemoglobin for nutrition, and be involved in invasion into host tissue. Therefore, cathepsin F serves as a potential target for parasitic disease immunodiagnosis, vaccine design and anti-parasite drug screening. This article reviews the structural characteristics and mechanisms of cathepsin F, and research advances on cathepsin F of parasitic helminths.

  20. Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

    Science.gov (United States)

    Železnik, Tajana Zajc; Kadin, Andrey; Turk, Vito; Dolenc, Iztok

    2015-09-18

    Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    International Nuclear Information System (INIS)

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  2. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  3. Antiviral activity and specific modes of action of bacterial prodigiosin against Bombyx mori nucleopolyhedrovirus in vitro.

    Science.gov (United States)

    Zhou, Wei; Zeng, Cheng; Liu, RenHua; Chen, Jie; Li, Ru; Wang, XinYan; Bai, WenWen; Liu, XiaoYuan; Xiang, TingTing; Zhang, Lin; Wan, YongJi

    2016-05-01

    Prodigiosin, the tripyrrole red pigment, is a bacterial secondary metabolite with multiple bioactivities; however, the antiviral activity has not been reported yet. In the present study, we found the antiviral activity of bacterial prodigiosin on Bombyx mori nucleopolyhedrovirus (BmNPV)-infected cells in vitro, with specific modes of action. Prodigiosin at nontoxic concentrations selectively killed virus-infected cells, inhibited viral gene transcription, especially viral early gene ie-1, and prevented virus-mediated membrane fusion. Under prodigiosin treatment, both progeny virus production and viral DNA replication were significantly inhibited. Fluorescent assays showed that prodigiosin predominantly located in cytoplasm which suggested it might interact with cytoplasm factors to inhibit virus replication. In conclusion, the present study clearly indicates that prodigiosin possesses significant antiviral activity against BmNPV.

  4. A broad survey of cathepsin K immunoreactivity in human neoplasms.

    Science.gov (United States)

    Zheng, Gang; Martignoni, Guido; Antonescu, Cristina; Montgomery, Elizabeth; Eberhart, Charles; Netto, George; Taube, Janis; Westra, William; Epstein, Jonathan I; Lotan, Tamara; Maitra, Anirban; Gabrielson, Edward; Torbenson, Michael; Iacobuzio-Donahue, Christine; Demarzo, Angelo; Shih, Ie Ming; Illei, Peter; Wu, T C; Argani, Pedram

    2013-02-01

    Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.

  5. New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

    DEFF Research Database (Denmark)

    Godiksen, Helene; Nielsen, Henrik Hauch

    2007-01-01

    A new and simple method to distinguish between cathepsin B and cathepsin L in crude extracts of herring (Clupea harengus) muscle has been established. An acid treatment of crude extracts (exposed to pH 3 for 5 min) activated a latent form of cathepsin L and inactivated cathepsin B. Furthermore......, in neutral crude extract, the hydrolysis of benzyloxycarbonyl-L-phenylalanyl-L-arginyl-4-methylcoumarine (Z-Phe-Arg-MCA) (cathepsin B and cathepsin L substrates) was between 0% and 15% of the hydrolysis of benzyloxycarbonyl-L-arginyl-L-arginyl-7-amino-4-methylcoumarine (Z-Arg-Arg-MCA; cathepsin B substrate......). Cathepsin B activity is measured in neutral extract using the specific cathepsin B substrate Z-Arg-Arg-MCA and cathepsin L activity is measured in acid-treated extract with Z-Phe-Arg-MCA as substrate. The specific cathepsin B inhibitor, CA-074, did not inhibit the Z-Arg-Arg-MCA significantly without...

  6. Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

    OpenAIRE

    Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

    2012-01-01

    Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of ...

  7. Cathepsin D SNP associated with increased risk of variant Creutzfeldt-Jakob disease

    Directory of Open Access Journals (Sweden)

    Sanchez-Juan Pascual

    2008-04-01

    Full Text Available Abstract Background Variant Creutzfeldt-Jakob disease (vCJD originally resulted from the consumption of foodstuffs contaminated by bovine spongiform encephalopathy (BSE material, with 163 confirmed cases in the UK to date. Many thousands are likely to have been exposed to dietary infection and so it is important (for surveillance, epidemic modelling, public health and understanding pathogenesis to identify genetic factors that may affect individual susceptibility to infection. This study looked at a polymorphism in the cathepsin D gene (refSNP ID: rs17571 previously examined in Alzheimer's disease (AD. Methods Blood samples taken from 110 vCJD patients were tested for the C-T base change, and genotype data were compared with published frequencies for a control population using multiple logistic regression. Results There was a significant excess of the cathepsin D polymorphism TT genotype in the vCJD cohort compared to controls. The TT genotype was found to have a 9.75 fold increase in risk of vCJD compared to the CT genotype and a 10.92 fold increase compared to the CC genotype. Conclusion This mutation event has been observed to alter the protease activity of the cathepsin D protein and has been linked to an increase in amyloid beta plaque formation in AD. vCJD neuropathology is characterised by the presence of amyloid plaques, formed from the prion protein, and therefore alterations in the amyloid processing activity of cathepsin D may affect the neuropathogenesis of this disease.

  8. Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease.

    Science.gov (United States)

    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2012-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Cathepsin E promotes pulmonary emphysema via mitochondrial fission.

    Science.gov (United States)

    Zhang, Xuchen; Shan, Peiying; Homer, Robert; Zhang, Yi; Petrache, Irina; Mannam, Praveen; Lee, Patty J

    2014-10-01

    Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. Elastin Degradation by Cathepsin V Requires Two Exosites*

    Science.gov (United States)

    Du, Xin; Chen, Nelson L. H.; Wong, Andre; Craik, Charles S.; Brömme, Dieter

    2013-01-01

    Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions. PMID:24121514

  11. The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

    Science.gov (United States)

    Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

    1995-10-06

    Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

  12. Evidence of recent interspecies horizontal gene transfer regarding nucleopolyhedrovirus infection of Spodoptera frugiperda.

    Science.gov (United States)

    Barrera, Gloria Patricia; Belaich, Mariano Nicolás; Patarroyo, Manuel Alfonso; Villamizar, Laura Fernanda; Ghiringhelli, Pablo Daniel

    2015-11-25

    Baculoviruses are insect-associated viruses carrying large, circular double-stranded-DNA genomes with significant biotechnological applications such as biological pest control, recombinant protein production, gene delivery in mammals and as a model of DNA genome evolution. These pathogens infect insects from the orders Lepidoptera, Hymenoptera and Diptera, and have high species diversity which is expressed in their diverse biological properties including morphology, virulence or pathogenicity. Spodoptera frugiperda (Lepidoptera: Noctuidae), the fall armyworm, represents a significant pest for agriculture in America; it is a host for baculoviruses such as the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (Colombia strain, genotype A) having been classified as a Group II alphabaculovirus making it a very attractive target for bioinsecticidal use. Genome analysis by pyrosequencing revealed that SfMNPV ColA has 145 ORFs, 2 of which were not present in the other sequenced genotypes of the virus (SfMNPV-NicB, SfMNPV-NicG, SfMNPV-19 and SfMNPV-3AP2). An in-depth bioinformatics study showed that ORF023 and ORF024 were acquired by a recent homologous recombination process between Spodoptera frugiperda and Spodoptera litura (the Oriental leafworm moth) nucleopolyhedroviruses. Auxiliary genes are numerous in the affected locus which has a homologous region (hr3), a repetitive sequence associated with genome replication which became lost in SfColA along with 1 ORF. Besides, the mRNAs associated with two acquired genes appeared in the virus' life-cycle during the larval stage. Predictive studies concerning the theoretical proteins identified that ORF023 protein would be a phosphatase involved in DNA repair and that the ORF024 protein would be a membrane polypeptide associated with cell transport. The SfColA genome was thus revealed to be a natural recombinant virus showing evidence of recent horizontal gene transfer between different baculovirus species occurring

  13. Characterization of the Bm61 of the Bombyx mori nucleopolyhedrovirus.

    Science.gov (United States)

    Shen, Hongxing; Chen, Keping; Yao, Qin; Zhou, Yang

    2009-07-01

    orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.

  14. Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes

    International Nuclear Information System (INIS)

    Oda, Kenzaburo; Luo, Yuqian; Yoshihara, Aya; Ishido, Yuko; Sekihata, Kengo

    2017-01-01

    Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways. - Highlights: • Follicular Tg increases cathepsin H mRNA and protein levels in rat thyroid cells. • Follicular Tg increases cathepsin H enzyme activity in rat thyroid cells. • After Tg stimulation cathepsin H co-localizes to lysosomes with follicular Tg. • Cathepsin H promotes hormone secretion by lysosome-mediated mechanisms.

  15. A bioavailable cathepsin S nitrile inhibitor abrogates tumor development.

    Science.gov (United States)

    Wilkinson, Richard D A; Young, Andrew; Burden, Roberta E; Williams, Rich; Scott, Christopher J

    2016-04-21

    Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models. Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors. We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis. In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

  16. Plasma levels of cathepsins L, K, and V and risks of abdominal aortic aneurysms

    DEFF Research Database (Denmark)

    Lv, Bing-Jie; Lindholt, Jes S; Wang, Jing

    2013-01-01

    Cathepsin L (CatL), cathepsin K (CatK), and cathepsin V (CatV) are potent elastases implicated in human arterial wall remodeling. Whether plasma levels of these cathepsins are altered in patients with abdominal aortic aneurysms (AAAs) remains unknown....

  17. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    International Nuclear Information System (INIS)

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-01-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  18. Detection of femtomole quantities of mature cathepsin K with zymography.

    Science.gov (United States)

    Li, Weiwei A; Barry, Zachary T; Cohen, Joshua D; Wilder, Catera L; Deeds, Rebecca J; Keegan, Philip M; Platt, Manu O

    2010-06-01

    Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Cathepsins are required for Toll-like receptor 9 responses

    International Nuclear Information System (INIS)

    Matsumoto, Fumi; Saitoh, Shin-ichiroh; Fukui, Ryutaroh; Kobayashi, Toshihiko; Tanimura, Natsuko; Konno, Kazunori; Kusumoto, Yutaka; Akashi-Takamura, Sachiko; Miyake, Kensuke

    2008-01-01

    Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9

  20. Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease.

    Science.gov (United States)

    Llorens, Franc; Thüne, Katrin; Sikorska, Beata; Schmitz, Matthias; Tahir, Waqas; Fernández-Borges, Natalia; Cramm, Maria; Gotzmann, Nadine; Carmona, Margarita; Streichenberger, Nathalie; Michel, Uwe; Zafar, Saima; Schuetz, Anna-Lena; Rajput, Ashish; Andréoletti, Olivier; Bonn, Stefan; Fischer, Andre; Liberski, Pawel P; Torres, Juan Maria; Ferrer, Isidre; Zerr, Inga

    2017-04-27

    Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrP Sc ). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca 2+ ) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.Here we describe the presence of massive regulation of Ca 2+ responsive genes in sCJD brain tissue, accompanied by two Ca 2+ -dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain proteins activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Additionally, Calpain 1 treatment enhances seeding activity of PrP Sc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons, although massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca 2+ homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

  1. Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

    NARCIS (Netherlands)

    Sun, X.; Chen, X.; Zhang, Z.; Wang, H.; Bianchi, F.J.J.A.; Peng, H.; Vlak, J.M.; Hu, Z.

    2002-01-01

    Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect.

  2. Field inactivation of wild-type and genetically modified Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in cotton

    NARCIS (Netherlands)

    Sun, X.; Sun, X.C.; Werf, van der W.; Vlak, J.M.; Hu, Z.H.

    2004-01-01

    Cotton bollworm (Helicoverpa armigera) is a serious pest on cotton in China. A specific baculovirus, H. armigera nucleopolyhedroviruses (HaSNPV) is used as a commercial biopesticide to control this pest. To improve the pesticidal properties, HaSNPV has been genetically engineered by both deleting

  3. The Operophtera brumata nucleopolyhedrovirus (OpbuNPV) represents an early, divergent lineage within genus Alphabaculovirus

    Science.gov (United States)

    Operophtera brumata nucleopolyhedrovirus (OpbuNPV) infects larvae of the winter moth, Operophtera brumata. As part of an effort to explore the pesticidal potential of OpbuNPV, an isolate of this virus from Massachusetts (USA), OpbuNPV-MA, was characterized by electron microscopy of OpbuNPV occlusio...

  4. Functional analysis of the Autographa californica nucleopolyhedrovirus IAP1 and IAP2

    NARCIS (Netherlands)

    Zeng, X.D.; Nan, F.; Liang, Ch.Y.; Song, J.H.; Wang, Q.; Vlak, J.M.; Chen, X.W.

    2009-01-01

    The Autographa californica nucleopolyhedrovirus (AcMNPV) contains three apoptosis suppressor genes: p35, iap1 and iap2. AcMNPV P35 functions as a pancaspase inhibitor, but the function of IAP1 and IAP2 has not been entirely resolved. In this paper, we analyze the function of IAP1 and IAP2 in detail.

  5. Diversity and biological activity of nucleopolyhedroviruses of the leafworm Spodoptera litura

    NARCIS (Netherlands)

    Ali, Ghulam

    2018-01-01

    Increased resistance of emerging cotton leafworm Spodoptera litura in Pakistan and elsewhere to chemical insecticides calls for an alternative method of control. Isolates of nucleopolyhedrovirus (NPVs) of S. litura (SpltNPV) were collected from infected larvae in different

  6. A hypothetical model of crossing Bombyx mori nucleopolyhedrovirus through its host midgut physical barrier.

    Directory of Open Access Journals (Sweden)

    Yang Cheng

    Full Text Available Bombyx mori nucleopolyhedrovirus (BmNPV is a primary pathogen of silkworm (B. mori that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.

  7. A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

    Science.gov (United States)

    Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

    2014-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV. PMID:25502928

  8. Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins

    Directory of Open Access Journals (Sweden)

    Jennifer M Rothberg

    2013-10-01

    Full Text Available One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8 affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D cultures. For these studies, we used 1 3D reconstituted basement membrane overlay cultures of human carcinomas, 2 live cell imaging assays to assess proteolysis, and 3 in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  9. Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

    Science.gov (United States)

    Chantree, Pathanin; Phatsara, Manussabhorn; Meemon, Krai; Chaichanasak, Pannigan; Changklungmoa, Narin; Kueakhai, Pornanan; Lorsuwannarat, Natcha; Sangpairoj, Kant; Songkoomkrong, Sineenart; Wanichanon, Chaitip; Itagaki, Tadashi; Sobhon, Prasert

    2013-09-01

    In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Excessive activity of cathepsin K is associated with cartilage defects in a zebrafish model of mucolipidosis II

    Directory of Open Access Journals (Sweden)

    Aaron C. Petrey

    2012-03-01

    The severe pediatric disorder mucolipidosis II (ML-II; also known as I-cell disease is caused by defects in mannose 6-phosphate (Man-6-P biosynthesis. Patients with ML-II exhibit multiple developmental defects, including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model of ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S and MMP-13 during early development. Unlike MMP-13, for which higher levels of protein were detected, the sustained activity of cathepsin K at later stages seemed to result from its abnormal processing and activation. Inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.

  11. Expression of Cathepsins B, D, and G in Isocitrate Dehydrogenase-Wildtype Glioblastoma

    Directory of Open Access Journals (Sweden)

    Sabrina P. Koh

    2017-05-01

    Full Text Available AimTo investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC subpopulations, we have previously characterized within isocitrate dehydogenase (IDH-wildtype glioblastoma (IDHWGB.Methods3,3-Diaminobezidine (DAB immunohistochemical (IHC staining for cathepsins B, D, and G, was performed on 4μm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric in situ hybridization (CISH were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance.ResultsCathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4+ and SALL4+ CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature.ConclusionThis study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more

  12. Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

    Science.gov (United States)

    Rojo, Liliana; Muhlia-Almazan, Adriana; Saborowski, Reinhard; García-Carreño, Fernando

    2010-11-01

    Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

  13. Histopathological analysis of cellular localization of cathepsins in abdominal aortic aneurysm wall.

    Science.gov (United States)

    Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Zimmermann, Alexander; Ockert, Stefan; Eckstein, Hans-Henning; Pelisek, Jaroslav

    2012-08-01

    An important feature of abdominal aortic aneurysm (AAA) is the destruction of vessel wall, especially elastin and collagen. Besides matrix metalloproteinases, cathepsins are the most potent elastolytic enzymes. The expression of cathepsins with known elastolytic and collagenolytic activities in the individual cells within AAA has not yet been determined. The vessel wall of 32 AAA patients and 10 organ donors was analysed by immunohistochemistry for expression of cathepsins B, D, K, L and S, and cystatin C in all cells localized within AAA. Luminal endothelial cells (ECs) of AAA were positive for cathepsin D and partially for cathepsins B, K and S. Endothelial cells of the neovessels and smooth muscle cells in the media were positive for all cathepsins tested, especially for cathepsin B. In the inflammatory infiltrate all cathepsins were expressed in the following pattern: B > D = S > K = L. Macrophages showed the highest staining intensity for all cathepsins. Furthermore, weak overall expression of cystatin C was observed in all the cells localized in the AAA with the exception of the ECs. There is markedly increased expression of the various cathepsins within the AAA wall compared to healthy aorta. Our data are broadly consistent with a role for cathepsins in AAA; and demonstrate expression of cathepsins D, B and S in phagocytic cells in the inflammatory infiltrate; and also may reveal a role for cathepsin B in lymphocytes. © 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.

  14. Upregulation of cathepsin S in psoriatic keratinocytes.

    Science.gov (United States)

    Schönefuss, Alexander; Wendt, Wiebke; Schattling, Benjamin; Schulten, Roxane; Hoffmann, Klaus; Stuecker, Markus; Tigges, Christian; Lübbert, Hermann; Stichel, Christine

    2010-08-01

    Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II-mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS-immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T- and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS-immunostaining is also detectable in keratinocytes. We show that cocultivation with T-cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T-cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II-associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.

  15. Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression

    NARCIS (Netherlands)

    Verbovšek, Urška; van Noorden, Cornelis J. F.; Lah, Tamara T.

    2015-01-01

    Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts,

  16. Identification of interleukin-8 converting enzyme as cathepsin L.

    Science.gov (United States)

    Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

    2003-06-26

    IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

  17. Expression and Localization of Cathepsins B, D, and G in Dupuytren’s Disease

    Directory of Open Access Journals (Sweden)

    Kirin Tan, MB ChB

    2018-02-01

    Conclusions:. Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

  18. Localization profile of Cathepsin L in the brain of African giant rat ...

    African Journals Online (AJOL)

    Cathepsins, are members of the papain superfamily of mammalian lysosomal cysteine proteases. Among others there are two prominent members with broad substrate specificity, these are cathepsin B and cathepsin L that are known to be involved in the process of intra- and extra-cellular protein degradation and turnover.

  19. Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17.

    Science.gov (United States)

    Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

    2012-12-01

    Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.

  20. Keefektifan Bahan Pelindung Alami Dalam Mempertahankan Infektivitas Spodoptera Exigua Nucleopolyhedrovirus (Senpv)' [the Effectiveness of Natural Protectant to Maintain the Spodoptera Exigua Nucleopolyhedrovirus (Senpv) Infectivity

    OpenAIRE

    Samsudin, Samsudin; Santoso, Teguh; Rauf, Aunu; Kusumah, Yayi Munara

    2011-01-01

    Spodoptera exigua nucleopolyhedrovirus (SeNPV) is a viral pathogen of onion caterpillar S. exigua with high pathogenicity. One of the major constraints to the use of SeNPV for biocontrol of onion caterpillar is its sensitivity to ultraviolet (UV) degradation. The purposes of this research were to determine the effect of sunlight exposure on the virulence of SeNPV and to find out the effective natural UV protectant to maintain the SeNPV virulence. The results showed that the sunlight radiation...

  1. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M.

    1989-01-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  2. Cathepsin-D And Tnf-α in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    T. Salman

    1996-01-01

    Full Text Available In a study of 34 normal healthy controls, 35 patients with urinary tract bilharziasis and 93 bladder cancer patients (62 of them are operable cases and 31 are non-operable ones, serum tumor necrosis factor alpha (TNF-α and cytosolic Cathepsin-D were estimated. Though both potential markers were elevated in bladder cancer patients, neither Cathepsin-D nor TNF-α showed associations of prognostic value since there were no positive correlations with tumor stages, grades or association of tumors with bilharzia ova or lymph node involvement.

  3. Genomic sequencing and analyses of HearMNPV—a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Tang Ping

    2012-08-01

    Full Text Available Abstract Background HearMNPV, a nucleopolyhedrovirus (NPV, which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy. HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV. To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. Methods The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the “partial filling-in” method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. Results Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B, but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome. Conclusions HearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.

  4. Biosafety of Recombinant and Wild Type Nucleopolyhedroviruses as Bioinsecticides

    Directory of Open Access Journals (Sweden)

    Bruce D. Hammock

    2007-06-01

    Full Text Available The entomopathogenic Autographa californica (Speyer nucleopolyhedrovirus (AcMNPV has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 1012 PIBs/feddan, feddan = 4,200 m2 and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field, AcMNPV, and SlNPV using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 μg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%, AcAaIT-field (1.2%, and SlNPV (4.0%. Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal

  5. Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

    Directory of Open Access Journals (Sweden)

    Nauder Faraday

    Full Text Available Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

  6. Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment

    NARCIS (Netherlands)

    Ortega-Gomez, Almudena; Salvermoser, Melanie; Rossaint, Jan; Pick, Robert; Brauner, Janine; Lemnitzer, Patricia; Tilgner, Jessica; de Jong, Renske J.; Megens, Remco T. A.; Jamasbi, Janina; Döring, Yvonne; Pham, Christine T.; Scheiermann, Christoph; Siess, Wolfgang; Drechsler, Maik; Weber, Christian; Grommes, Jochen; Zarbock, Alexander; Walzog, Barbara; Soehnlein, Oliver

    2016-01-01

    Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the

  7. Structure of the periodontium in cathepsin C-deficient mice

    NARCIS (Netherlands)

    de Haar, Susanne F.; Tigchelaar-Gutter, Wikky; Everts, Vincent; Beertsen, Wouter

    2006-01-01

    Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency

  8. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

  9. Complex modulation of peptidolytic activity of cathepsin D by sphingolipids

    Czech Academy of Sciences Publication Activity Database

    Žebrakovská, Iva; Máša, Martin; Srp, Jaroslav; Horn, Martin; Vávrová, K.; Mareš, Michael

    2011-01-01

    Roč. 1811, č. 12 (2011), s. 1097-1104 ISSN 1388-1981 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506 Keywords : sphingolipid * phospholipid * inhibition * activation * cathepsin D * enzyme regulation Subject RIV: CE - Biochemistry Impact factor: 5.269, year: 2011

  10. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. Copyright © 2016. Published by Elsevier B.V.

  11. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  12. Cathepsin D and Its Prognostic Value in Neuroepithelial Brain Tumors

    OpenAIRE

    Pigac, Biserka; Dmitrović, Branko; Marić, Svjetlana; Mašić, Silvija

    2012-01-01

    Expression of Cathepsin D (Cath D) in some primary neuroepithelial brain tumors and its prognostic value were studied. The research included 65 samples of human primary neuroepithelial brain tumors. There were 50 glial tumors (10 diffuse astrocytomas (DA), 15 anaplastic astrocytomas (AA), 25 glioblastomas (GB), 15 embryonic tumors (15 medulloblastomas (MB) as well as 5 samples of normal brain tissue. Immunohistochemical method was applied to monitor diffuse positive reaction in the cytoplasm ...

  13. Structure of a Kunitz-type potato cathepsin D inhibitor

    Czech Academy of Sciences Publication Activity Database

    Guo, J.; Erskine, P. T.; Coker, A. R.; Wood, S. P.; Cooper, J. B.; Mareš, Michael; Baudyš, Miroslav

    2015-01-01

    Roč. 192, č. 3 (2015), s. 554-560 ISSN 1047-8477 R&D Projects: GA ČR GA15-18929S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : potato cathepsin D inhibitor * Kunitz-type protease inhibitor * protein X-ray structure * reactive-site loop * docking Subject RIV: CE - Biochemistry Impact factor: 2.570, year: 2015

  14. Cathepsin E deficiency impairs autophagic proteolysis in macrophages.

    Directory of Open Access Journals (Sweden)

    Takayuki Tsukuba

    Full Text Available Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/- mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR, Akt, and extracellular signal-related kinase (ERK. Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/- macrophages showed increased reactive oxygen species (ROS production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

  15. Betabaculovirus F proteins showed different efficiencies when rescuing the infectivity of gp64-null Autographa californica nucleopolyhedrovirus

    NARCIS (Netherlands)

    Yin, F.; Wang, M.; Ying, T.; Deng, F.; Vlak, J.M.; Hu, Z.; Wang, H.

    2013-01-01

    The Agrotis segetum granulovirus (AgseGV) F protein was previously identified as the first betabaculovirus F protein with functional homology to Autographa californica nucleopolyhedrovirus (AcMNPV) GP64. In the current study, F proteins from Xestia c-nigrum granulovirus (XecnGV), Cydia pomonella

  16. P143 proteins from heterologous nucleopolyhedroviruses induce apoptosis in BM-N cells derived from the silkworm Bombyx mori.

    Science.gov (United States)

    Hamajima, Rina; Kobayashi, Michihiro; Ikeda, Motoko

    2017-04-02

    We previously demonstrated that ribosomal RNA (rRNA) of Bombyx mori BM-N cells is rapidly degraded upon infection with heterologous nucleopolyhedroviruses (NPVs), including Autographa californica multiple NPV (AcMNPV), Hyphantria cunea MNPV, Spodoptera exigua MNPV and S. litura MNPV, and that this response is triggered by viral P143 proteins. The transient expression of P143 proteins from heterologous NPVs was also shown to induce apoptosis and caspase-3-like protease activation in BM-N cells. In the present study, we conducted a transient expression assay using BM-N cells expressing mutant AcMNPV P143 (Ac-P143) proteins and demonstrated that five amino acid residues cooperatively participate in Ac-P143 protein-triggered apoptosis of BM-N cells. Notably, these five residues were previously shown to be required for triggering rRNA degradation in BM-N cells. As rRNA degradation in BM-N cells does not result from apoptosis, the present results suggest that Ac-P143-triggered rRNA degradation is the upstream signal for apoptosis induction in BM-N cells. We further showed that P143 protein-triggered apoptosis does not occur in S. frugiperda Sf9 or Lymantria dispar Ld652Y cells, indicating that apoptosis induction by heterologous P143 proteins is a BM-N cell-specific response. In addition, the observed induction of apoptosis in BM-N cells was found to be mediated by activation of the initiator caspase Bm-Dronc. Taken together, these results suggest that BM-N cells evolved a unique antiviral system that recognizes heterologous NPV P143 proteins to induce rRNA degradation and caspase-dependent apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population

    Science.gov (United States)

    Barrera, Gloria; Williams, Trevor; Villamizar, Laura; Caballero, Primitivo; Simón, Oihane

    2013-01-01

    A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host. PMID:24116220

  18. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    Science.gov (United States)

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Transmission of Different Nucleopolyhedroviruses by Two Ectoparasitoids – Bracon hebetor Say (Hymenoptera: Braconidae and Euplectrus plathypenae (Howard (Hymenoptera: Eulophidae

    Directory of Open Access Journals (Sweden)

    Emanouela E. Stoianova

    2010-01-01

    Full Text Available The transmission of nucleopolyhedroviruses (NPVs of Autographa gamma (AgNPV,Mamestra brassicae (MbNPV, Lacanobia oleraceae (LoNPV, Helicoverpa armigera (HaNPVand Xantia c-nigrum (XnNPV to their relevant larvae by the ectoparasitoid Bracon hebetorand the transmission of the multiple-enveloped NPVs of Spodoptera exigua (SeMNPV andSpodoptera frugiperda (SfMNPV by the ectoparasitoid Euplectrus plathypenae was examined.Two methods of contamination of the both parasitoids (exposure to infected hostsand total body surface and two subsequent transmissions of the viruses by Bracon hebetorto healthy hosts were tested. The results showed that both parasitoids were capable tobe mechanical vectors of the tested NPVs. Every Bracon hebetor female was able to transmitsubsequently twice the virus in 27% to 52.2% of the five Noctuidae species by preliminaryexposing to infected larvae. The second method of contamination (applying virus suspensionto the total body surface of the parasitoid was also efficient causing virus infection inbetween 29.4% and 54.15% of the larvae.The parasitoid E. plathypenae transmited the virus from infected to noninfected larvaein 20% and 25.57% of the S. frugiperda and S. exigua larvae, and 6.43% and 11.10%, respectivelyof them died from the virus infection. The same observation was established by thesecond method of contamination – respectively 33.33% and 40% infection and between13.23% and 16.67% mortality. The mortality of all tested larvae exposed to virus contaminated parasitoids was higherwhen the parasitoid entire body surface had been artificially contaminated with the virusthan when the parasitoid itself was previously allowed to oviposit the larvae.

  20. Entry into Midgut Epithelial Cells is a Key Step in the Selection of Genotypes in a Nucleopolyhedrovirus

    Institute of Scientific and Technical Information of China (English)

    Gabriel Clavijo; Trevor Williams; Delia Mu(n)oz; Miguel L(o)pez-Ferber; Primitivo Caballero

    2009-01-01

    An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.

  1. Deletion genotypes reduce occlusion body potency but increase occlusion body production in a Colombian Spodoptera frugiperda nucleopolyhedrovirus population.

    Directory of Open Access Journals (Sweden)

    Gloria Barrera

    Full Text Available A Colombian field isolate (SfCOL-wt of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J. SfCOL-A was the most prevalent (71±2%; mean ± SE showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F. Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.

  2. The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

    Science.gov (United States)

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

    2013-10-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

  3. Cysteine cathepsins B and X promote epithelial-mesenchymal transition of tumor cells.

    Science.gov (United States)

    Mitrović, Ana; Pečar Fonović, Urša; Kos, Janko

    2017-09-01

    Cathepsins B and X are lysosomal cysteine carboxypeptidases suggested as having a redundant role in cancer. They are involved in a number of processes leading to tumor progression but their role in the epithelial-mesenchymal transition (EMT) remains unknown. We have investigated the contribution of both cathepsins B and X in EMT using tumor cell lines differing in their expression of epithelial and mesenchymal markers and cell morphology. Higher levels of both cathepsins are shown to promote EMT and are associated with the mesenchymal-like cell phenotype. Moreover, simultaneous knockdown of the two peptidases triggers a reverse, mesenchymal to epithelial transition. Of the two cathepsins, cathepsin B appears to be the stronger promotor of EMT. Furthermore, we evaluated the involvement of cathepsin B and X in the transforming growth factor-β1 (TGF-β1) signaling pathway, one of the key signaling mechanisms triggering EMT in cancer. In MCF-7 cells the expression of cathepsin B was shown to depend on their activation with TGF-β1 while, for cathepsin X, a TGF-β1 independent mechanism of induction during EMT is indicated. EMT is thus shown to be another mechanism linking cathepsins B and X with tumor progression. With silencing of their expression or inhibition of enzymatic activity, the tumor cells could be reverted to less aggressive epithelial-like phenotype. Copyright © 2017 Elsevier GmbH. All rights reserved.

  4. Cysteine Cathepsins as Regulators of the Cytotoxicity of NK and T Cells

    Science.gov (United States)

    Perišić Nanut, Milica; Sabotič, Jerica; Jewett, Anahid; Kos, Janko

    2014-01-01

    Cysteine cathepsins are lysosomal peptidases involved at different levels in the processes of the innate and adaptive immune responses. Some, such as cathepsins B, L, and H are expressed constitutively in most immune cells. In cells of innate immunity they play a role in cell adhesion and phagocytosis. Other cysteine cathepsins are expressed more specifically. Cathepsin X promotes dendritic cell maturation, adhesion of macrophages, and migration of T cells. Cathepsin S is implicated in major histocompatibility complex class II antigen presentation, whereas cathepsin C, expressed in cytotoxic T lymphocytes and natural killer (NK) cells, is involved in processing pro-granzymes into proteolytically active forms, which trigger cell death in their target cells. The activity of cysteine cathepsins is controlled by endogenous cystatins, cysteine protease inhibitors. Of these, cystatin F is the only cystatin that is localized in endosomal/lysosomal vesicles. After proteolytic removal of its N-terminal peptide, cystatin F becomes a potent inhibitor of cathepsin C with the potential to regulate pro-granzyme processing and cell cytotoxicity. This review is focused on the role of cysteine cathepsins and their inhibitors in the molecular mechanisms leading to the cytotoxic activity of T lymphocytes and NK cells in order to address new possibilities for regulation of their function in pathological processes. PMID:25520721

  5. The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Md Zahidul Islam Pranjol

    2015-11-01

    Full Text Available Epithelial ovarian cancer (EOC is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

  6. The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer.

    Science.gov (United States)

    Pranjol, Md Zahidul Islam; Gutowski, Nicholas; Hannemann, Michael; Whatmore, Jacqueline

    2015-11-20

    Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

  7. Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

    International Nuclear Information System (INIS)

    Andrade, Sheila Siqueira; Gouvea, Iuri Estrada; Silva, Mariana Cristina C.; Castro, Eloísa Dognani; Paula, Cláudia A. A. de; Okamoto, Debora; Oliveira, Lilian; Peres, Giovani Bravin; Ottaiano, Tatiana; Facina, Gil; Nazário, Afonso Celso Pinto; Campos, Antonio Hugo J. F. M.; Paredes-Gamero, Edgar Julian; Juliano, Maria; Silva, Ismael D. C. G. da; Oliva, Maria Luiza V.; Girão, Manoel J. B. C.

    2016-01-01

    Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and −4 are highly expressed, but PAR-3 shows low expression and unclear functions. Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and −4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells. The online version of this article (doi:10.1186/s12885-016-2203-7) contains supplementary material, which is available to authorized users

  8. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  9. [Effect of cryoconservation of platelets on polyamine level and cathepsin D activity].

    Science.gov (United States)

    Kovtunova, M E; Selezneva, O M; Vorozhtsova, S I; Kunof, V K

    1992-01-01

    Alterations in activity of cathepsin D as well as in content of polyamines spermine, spermidine, x-fraction (apparently, acetyl spermidine) and putrescine were studied in blood platelets depending on conditions of conservation. The enzyme activity and content of polyamines correlated directly with duration of the cell concentrates storage. Cathepsin D and polyamines appear to be involved in responses to stress adaptation.

  10. Fibrinogen and fibrin are novel substrates for Fasciola hepatica cathepsin L peptidases

    NARCIS (Netherlands)

    Mebius, Mirjam M.; Op Heij, Jody M J; Tielens, Aloysius G.M.; de Groot, Philip G; Urbanus, Rolf T; van Hellemond, Jaap J.

    2018-01-01

    Cathepsin peptidases form a major component of the secreted proteins of the blood-feeding trematodes Fasciola hepatica and Schistosoma mansoni. These peptidases fulfill many functions, from facilitating infection to feeding and immune evasion. In this study, we examined the Fasciola cathepsin L

  11. The development and characterization of an ELISA specifically detecting the active form of cathepsin K

    DEFF Research Database (Denmark)

    Sun, S; Karsdal, M A; Bay-Jensen, A C

    2013-01-01

    Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or anky...

  12. Serum cathepsin H as a potential prognostic marker in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Schweiger, A; Christensen, Ib Jarle; Nielsen, Hans Jørgen

    2005-01-01

    Cathepsin H is a lysosomal cysteine protease that may participate in tumor progression. In order to evaluate its potential as a prognostic marker, its protein levels were measured by ELISA in preoperative sera from 324 patients with colorectal cancer. The level of cathepsin H was significantly...... increased in patient sera, the median level was 8.4 ng/mL versus 2.1 ng/mL in 90 healthy blood donors (p CEA). In survival analysis...... a significant difference was found between the group of patients with low cathepsin H (first tertile) who had a poor prognosis and the remaining patients (p = 0.03). The risk of patients was further stratified when cathepsin H levels were combined with CEA. Patients with high CEA and low cathepsin H had...

  13. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

    DEFF Research Database (Denmark)

    Parker, Erica N; Song, Jiangli; Kishore Kumar, G D

    2015-01-01

    selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5μ......Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from......) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L...

  14. Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies.

    Science.gov (United States)

    Meemon, Krai; Sobhon, Prasert

    2015-08-01

    Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is one of the most neglected tropical zoonotic diseases. One sustainable control strategy against these infections is the employment of vaccines that target proteins essential for parasites' invasion and nutrition acquiring processes. Cathepsin proteases are the most abundantly expressed proteins in Fasciola spp. that have been tested successfully as vaccines against fasciolosis in experimental as well as large animals because of their important roles in digestion of nutrients, invasion, and migration. Specifically, juvenile-specific cathepsin proteases are the more effective vaccines because they could block the invasion and migration of juvenile parasites whose immune evasion mechanism has not yet been fully developed. Moreover, because of high sequence similarity and identity of cathepsins from juveniles with those of adults, the vaccines can attack both the juvenile and adult stages. In this article, the characteristics and vaccine potentials of juvenile-specific cathepsins, i.e., cathepsins L and B, of Fasciola spp. were reviewed.

  15. Prediction of Aggressive Human Prostate Cancer by Cathepsin B

    Science.gov (United States)

    2008-03-01

    Cancer Res 2004;10(12 Pt 1):4118-4124. 28. Munoz E, Gomez F, Paz JI, Casado I, Silva JM, Corcuera MT, Alonso MJ. Ki-67 immunolabeling in pre...detected prostate cancer. J Pathol 2002;197(2):148-154. 34. Claudio PP, Zamparelli A, Garcia FU, Claudio L, Ammirati G, Farina A, Bovicelli A, Russo G...JA. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev 2006;20(5):543-556. 47. Fernandez PL, Farre X, Nadal A

  16. Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

    Science.gov (United States)

    Wongwairot, Sirima; Kueakhai, Pornanan; Changklungmoa, Narin; Jaikua, Wipaphorn; Sansri, Veerawat; Meemon, Krai; Songkoomkrong, Sineenart; Riengrojpitak, Suda; Sobhon, Prasert

    2015-01-01

    Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

  17. Autographa californica multiple nucleopolyhedrovirus PK-1 is essential for nucleocapsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Changyong, E-mail: cyliang@yzu.edu.cn [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Li, Min; Dai, Xuejuan; Zhao, Shuling; Hou, Yanling; Zhang, Yongli; Lan, Dandan [College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009 (China); Wang, Yun; Chen, Xinwen [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2013-09-01

    PK-1 (Ac10) is a baculovirus-encoded serine/threonine kinase and its function is unclear. Our results showed that a pk-1 knockout AcMNPV failed to produce infectious progeny, while the pk-1 repair virus could rescue this defect. qPCR analysis demonstrated that pk-1 deletion did not affect viral DNA replication. Analysis of the repaired recombinants with truncated pk-1 mutants demonstrated that the catalytic domain of protein kinases of PK-1 was essential to viral infectivity. Moreover, those PK-1 mutants that could rescue the infectious BV production defect exhibited kinase activity in vitro. Therefore, it is suggested that the kinase activity of PK-1 is essential in regulating viral propagation. Electron microscopy revealed that pk-1 deletion affected the formation of normal nucleocapsids. Masses of electron-lucent tubular structures were present in cell transfected with pk-1 knockout bacmid. Therefore, PK-1 appears to phosphorylate some viral or cellular proteins that are essential for DNA packaging to regulate nucleocapsid assembly. - Highlights: • A pk-1 knockout AcMNPV failed to produce infectious progeny. • The pk-1 deletion did not affect viral DNA replication. • The catalytic domain of protein kinases (PKc) of PK-1 was essential to viral infectivity. • The kinase activity of PK-1 is essential in regulating viral propagation. • PK-1 appears to phosphorylate some viral proteins that are essential for DNA packaging to regulate nucleocapsid assembly.

  18. BM61 of Bombyx mori nucleopolyhedrovirus: its involvement in the egress of nucleocapsids from the nucleus.

    Science.gov (United States)

    Shen, Hongxing; Chen, Keping

    2012-04-05

    All lepidopteran baculovirus genomes sequenced encode a homolog of the Bombyx mori nucleopolyhedrovirus orf61 gene (Bm61). To determine the role of Bm61 in the baculoviral life cycle, we constructed a Bm61 knockout virus and characterized it in cells. We observed that the Bm61 deletion bacmid led to a defect in production of infectious budded virus (BV). Quantitative PCR analysis of BV in the media culturing the transfected cell indicated that BV was not produced due to Bm61 deletion. Electron microscope analysis showed that in the knockout of Bm61, nucleocapsids were not transported from the nucleus to the cytoplasm. From these results we concluded that BM61 is required in the BV pathway for the egress of nucleocapsids from the nucleus to the cytoplasm. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

    Directory of Open Access Journals (Sweden)

    Yang Qiong

    Full Text Available ABSTRACT Bombyx mori nucleopolyhedrovirus (BmNPV disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM. Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM. The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.

  20. Low-Cost Method to Monitor Patient Adherence to HIV Antiretroviral Therapy Using Multiplex Cathepsin Zymography.

    Science.gov (United States)

    Platt, Manu O; Evans, Denise; Keegan, Philip M; McNamara, Lynne; Parker, Ivana K; Roberts, LaDeidra M; Caulk, Alexander W; Gleason, Rudolph L; Seifu, Daniel; Amogne, Wondwossen; Penny, Clement

    2016-01-01

    Monitoring patient adherence to HIV antiretroviral therapy (ART) by patient survey is inherently error prone, justifying a need for objective, biological measures affordable in low-resource settings where HIV/AIDS epidemic is highest. In preliminary studies conducted in Ethiopia and South Africa, we observed loss of cysteine cathepsin activity in peripheral blood mononuclear cells of HIV-positive patients on ART. We optimized a rapid protocol for multiplex cathepsin zymography to quantify cysteine cathepsins, and prospectively enrolled 350 HIV-positive, ART-naïve adults attending the Themba Lethu Clinic, Johannesburg, South Africa, to test if suppressed cathepsin activity could be a biomarker of ART adherence (103 patients were included in final analysis). Poor adherence was defined as detectable viral load (>400 copies/ml) or simplified medication adherence questionnaire, 4-6 months after ART initiation. 86 % of patients with undetectable viral loads after 6 months were cathepsin negative, and cathepsin-positive patients were twice as likely to have detectable viral loads (RR 2.32 95 % CI 1.26-4.29). Together, this demonstrates proof of concept that multiplex cathepsin zymography may be an inexpensive, objective method to monitor patient adherence to ART. Low cost of this electrophoresis-based assay makes it a prime candidate for implementation in resource-limited settings.

  1. Low cost method to monitor patient adherence to HIV antiretroviral therapy using multiplex cathepsin zymography

    Science.gov (United States)

    Platt, Manu O.; Evans, Denise; Keegan, Philip M.; McNamara, Lynne; Parker, Ivana K.; Roberts, LaDeidra M.; Caulk, Alexander W.; Gleason, Rudolph L.; Seifu, Daniel; Amogne, Wondwossen; Penny, Clement

    2015-01-01

    Monitoring patient adherence to HIV antiretroviral therapy (ART) by patient survey is inherently error-prone, justifying a need for objective, biological measures affordable in low resource settings where HIV/AIDS epidemic is highest. In preliminary studies conducted in Ethiopia and South Africa, we observed loss of cysteine cathepsin activity in peripheral blood mononuclear cells (PBMCs) of HIV-positive patients on ART. We optimized a rapid protocol for multiplex cathepsin zymography to quantify cysteine cathepsins, and prospectively enrolled 350 HIV-positive, ART naïve adults attending the Themba Lethu Clinic, Johannesburg, South Africa, to test if suppressed cathepsin activity could be a biomarker of ART adherence (103 patients were included in final analysis). Poor adherence was defined as detectable viral load (>400 copies/ml) or simplified medication adherence questionnaire (SMAQ), 4–6 months after ART initiation. 86% of patients with undetectable viral loads after 6 months were cathepsin negative, and cathepsin positive patients were twice as likely to have detectable viral loads (RR 2.32 95% CI 1.26–4.29). Together, this demonstrates proof of concept that multiplex cathepsin zymography may be an inexpensive, objective method to monitor patient adherence to ART. Low cost of this electrophoresis based assay makes it a prime candidate for implementation in resource limited settings. PMID:26589706

  2. Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus

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    Asami Yoshida

    2015-10-01

    Full Text Available An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s, such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

  3. Cathepsin L of Triatoma brasiliensis (Reduviidae, Triatominae): sequence characterization, expression pattern and zymography.

    Science.gov (United States)

    Waniek, Peter J; Pacheco Costa, Juliana E; Jansen, Ana M; Costa, Jane; Araújo, Catarina A C

    2012-01-01

    Triatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions.

    Science.gov (United States)

    Jerič, Barbara; Dolenc, Iztok; Mihelič, Marko; Klarić, Martina; Zavašnik-Bergant, Tina; Gunčar, Gregor; Turk, Boris; Turk, Vito; Stoka, Veronika

    2013-10-01

    The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries

    Science.gov (United States)

    Glusa, Erika; Adam, Christine

    2001-01-01

    Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2α (3 μM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC50=9.64±0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC50=9.23±0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 μM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC50=0.043 μg ml−1), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC50=0.14 μg ml−1) of the cathepsin G effect, whereas neither indomethacin (3 μM) nor the thrombin inhibitor hirudin (5 ATU ml−1) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC50=0.11 μg ml−1), heparin (IC50=0.48 μg ml−1) and suramin (IC50=1.85 μg ml−1) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like

  6. Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis

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    Zhou Chenhui

    2011-07-01

    Full Text Available Abstract Background Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB and to investigate its diagnostic value for human helminthiases. Results The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases. Conclusions Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.

  7. Purification and characterization of cathepsin B from the skeletal muscles of agama stellio stellio

    International Nuclear Information System (INIS)

    El-Jassabi, S.; Abu-Ghalyun, Y.

    1997-01-01

    1. Cathepsin b from the muscles of Jordanian lizard Agama stellio stellio was purified to homogeneity by a series of column chromatography on DEAE-sephadex, thio propyl sepharose and sephadex G-100 2. The molecular weight of cathepsin B isolated was to be 31800 dalton by using SDS-PAGE, and 33000 dalton by gel filtration, and its isoelectric point was measured to be 4.2 by isoelectric focusing. 3. Cathepsin B had ph optimum of 5.5, required a thiol-reducing reagent for activation and was inhibited by thiol-protease inhibitors. 4. The Km and K cat values for Z-Phe-Arg-Mca were determined to be 0.161mM and 238 S -1 . 5. Cathepsin B acted on oligopeptide substrates mainly as di peptidyl carboxypeptidase. (authors). 22 refs., 3 tabs., 2 figs

  8. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: david.kirsch@duke.edu [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  9. Serum and saliva levels of cathepsin L in patients with acute coronary syndrome.

    Science.gov (United States)

    Mirzaii-Dizgah, Iraj; Riahi, Esmail

    2011-03-01

    Coronary artery disease (CAD) is the major cause of death nearly all over the world, and accurate and rapid diagnosis of CAD is of major medical and economic importance. The aim of this study was to evaluate the serum and saliva levels of cathepsin L in patients with acute coronary syndrome (ACS). In a cross-sectional study, 39 patients with ACS and 28 with controls were recruited to the study, and cathepsin L levels were measured in serum, resting saliva, and stimulated saliva obtained 12 and 24 h after the onset of ACS by ELISA method. Statistical analyses of Fisher's exact test, the Student's t-test or Kruskal-Wallis test were performed. Stimulated saliva cathepsin L levels in patients with ACS 12 hours but not 24 hours after admission showed significant decrease compared with that in control subjects. However, there were no significant differences in serum and unstimulated saliva cathepsin L levels between groups. Serum and saliva levels of cathepsin L remain unchanged in patients with ACS and hence may not be a promising factor in CAD risk assessment. It seems that serum and saliva cathepsin L may not be a good biomarker for CHD. CAD: Coronary artery disease, ACS: Acute coronary syndrome, CHD: Coronary heart disease, EU: Emergency unit, MI: Myocardial infarction. Cathepsin L, Acute coronary syndrome, Resting saliva, Stimulated saliva. How to cite this article: Mirzaii-Dizgah I, Riahi E. Serum and Saliva Levels of Cathepsin L in Patients with Acute Coronary Syndrome. J Contemp Dent Pract 2011;12(2):114-119.

  10. Gelatin Zymography Using Leupeptin for the Detection of Various Cathepsin L Forms.

    Science.gov (United States)

    Hashimoto, Yoko

    2017-01-01

    Zymography is a highly sensitive method to assess the activities as well as molecular weights of enzymes in crude biological fluids and tissue extracts. Cathepsin L is a lysosomal cysteine proteinase that is optimally active at slightly acidic pH and is highly unstable in alkaline solutions such as electrode buffer (pH 8.3). Large amounts of cathepsin L are secreted by various cancer cells, where it promotes invasion and metastasis. Leupeptin is a tight-binding inhibitor of cysteine proteinases, and its complex with cathepsin L is stable in alkaline solutions. Moreover, leupeptin can be easily removed from the complex because it is a reversibly binding inhibitor. In addition, leupeptin is too small to influence the electrode migration distance of the complex with cathepsin L on a sodium dodecyl sulfate-polyacrylamide gel. Here, a novel gelatin zymography technique that employs leupeptin to detect pro-, intermediate, and mature cathepsin L forms on the basis of their gelatinolytic activities is described. Further, the differences in the glycosylation, phosphorylation, and processing statuses of lysosomal and secreted cathepsin L forms isolated from cultured HT 1080 cells are demonstrated using this method.

  11. [Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

    Science.gov (United States)

    Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

    2013-01-01

    To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

  12. Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

    Science.gov (United States)

    Pečar Fonović, Urša; Jevnikar, Zala; Rojnik, Matija; Doljak, Bojan; Fonović, Marko; Jamnik, Polona; Kos, Janko

    2013-01-01

    Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies. PMID:23326535

  13. Profilin 1 as a target for cathepsin X activity in tumor cells.

    Directory of Open Access Journals (Sweden)

    Urša Pečar Fonović

    Full Text Available Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

  14. Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer.

    LENUS (Irish Health Repository)

    Sullivan, Shane

    2012-02-01

    Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

  15. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

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    Zhanqi Dong

    2018-02-01

    Full Text Available The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV immediate early-1 (ie-1 gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-/sgRNA(-, Cas9(+/sgRNA(-, Cas9(-/sgRNA(+, and Cas9(+/sgRNA(+. We demonstrated that the Cas9(+/sgRNA(+ transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+/sgRNA(+ transgenic lines shows that the median lethal dose (LD50 is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+/sgRNA(+ transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

  16. Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

    Science.gov (United States)

    Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

    2018-01-01

    The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

  17. Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates.

    Science.gov (United States)

    Bouwer, Gustav; Nardini, Luisa; Duncan, Frances D

    2009-04-01

    To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO(2) production (VCO(2)), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD(25) or LD(75) survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO(2) values of larvae that survived inoculation (0.0288mlh(-1)), the uninoculated controls (0.0250mlh(-1)), and the larvae that did not survive inoculation (0.0199mlh(-1)) differed significantly from one another. At 4dpi, the VCO(2) of the uninoculated controls were significantly lower than the VCO(2) of inoculation survivors, but significantly higher than the VCO(2) of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.

  18. Genome sequence and organization of a nucleopolyhedrovirus isolated from the smaller tea tortrix, Adoxophyes honmai

    International Nuclear Information System (INIS)

    Nakai, Madoka; Goto, Chie; Kang, Won Kyung; Shikata, Masamitsu; Luque, Teresa; Kunimi, Yasuhisa

    2003-01-01

    Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) has a distinctive pathology in A. honmai larvae, killing the host more slowly than other NPVs. To further understand the pathology of AdhoNPV, its genome was completely sequenced and compared with those of other baculoviruses. The AdhoNPV genome is 113,220 bp, with a G + C content of 35.6%. It contains 125 putative open reading frames (ORFs), of which 8 are unique to AdhoNPV, and 4 homologous regions. The other 117 ORFs display similarity to previously characterized baculovirus genes involved in early and late gene expression, DNA replication, and structural and auxiliary functions. The phylogenetic position of AdhoNPV, in relation to 15 other baculoviruses whose genomes have been completely sequenced, was assessed by three different analyses: gene sequence, gene order, and gene content. Although gene content analysis failed to support the group II NPVs, phylogenetic trees based on gene sequence and gene order showed AdhoNPV to be closely related to the group II NPVs

  19. Bm65 is essential for the propagation of Bombyx mori nucleopolyhedrovirus.

    Science.gov (United States)

    Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Feng, Fan; Yuan, Yi; Chen, Keping

    2013-01-01

    Orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes an unknown 104-amino acid protein. In the present study, we have shown the role of Bm65 in the baculovirus life cycle. 5'-RACE analysis showed that the transcription start site of Bm65 was 14 nucleotides upstream of the start codon ATG. The transcription profile of Bm65 was detected from 6 to 72 h postinfection (p. i.) by RT-PCR. A Bm65-knockout bacmid was constructed by homologous recombination to characterize the role of Bm65 in viral life cycle. Fluorescence microscopy showed that Bm65-knockout virus was unable to generate infectious budded virus in BmN cells. Furthermore, quantitative real-time PCR analysis demonstrated that Bm65 deletion did not affect the viral DNA replication. To conclude, Bm65 is essential for the propagation of BmNPV, but is unnecessary for the replication of viral DNA.

  20. Sublethal dose of phoxim and Bombyx mori nucleopolyhedrovirus interact to elevate silkworm mortality.

    Science.gov (United States)

    Gu, ZhiYa; Li, FanChi; Hu, JingSheng; Ding, Chao; Wang, Chaoqian; Tian, JiangHai; Xue, Bin; Xu, KaiZun; Shen, WeiDe; Li, Bing

    2017-03-01

    Silkworm (Bombyx mori) is an economically important insect. It is relatively less resistant to certain chemicals and environment exposures such as pesticides and pathogens. After pesticide exposures, the silkworms are more susceptible to microbial infections. The mechanism underlying the susceptibility might be related to immune response and oxidative stress. A sublethal dose of phoxim combined with Bombyx mori nucleopolyhedrovirus (BmNPV) elevated the silkworm mortality at 96 h. We found a higher content of H 2 O 2 and increased levels of genes related to oxidative stress and immune response after treatment with a sublethal dose of phoxim for 24 h or 48 h. However, such response decreased with longer pesticide treatment. Mortality increased by 44% when B. mori was exposed to combined treatment with BmNPV and phoxim rather than BmNPV alone. The level of examined immune-related and oxidative-stress-related genes significantly decreased in the combined treatment group compared with the BmNPV group. Our results indicated that, with long-term exposure to pesticides such as OPs, even at sublethal dose, the oxidative stress response and immune responses in silkworm were inhibited, which may lead to further immune impairment and accumulation of oxidative stress, resulting in susceptibility to the virus and harm to the silkworm. Our study provided insights for understanding the susceptibility to pathogen after pesticide exposures, which may promote the development of better pesticide controls to avoid significant economic losses. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  1. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    International Nuclear Information System (INIS)

    Kokusho, Ryuhei; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu

    2016-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  2. Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus

    International Nuclear Information System (INIS)

    Nakazawa, Hiroshi; Tsuneishi, Eiko; Ponnuvel, Kangayam M.; Furukawa, Seiichi; Asaoka, Ai; Tanaka, Hiromitsu; Ishibashi, Jun; Yamakawa, Minoru

    2004-01-01

    A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. The molecular mass of this protein was 24 271 Da. Partial N-terminal amino acid sequence of the protein was determined and cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 94% identity with B. mori serine protease so the protein was designated B. mori serine protease-2 (BmSP-2). Analysis of BmSP-2 gene expression showed that this gene is expressed in the midgut but not in other tissues. In addition, BmSP-2 gene was shown to not be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that BmSP-2, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection

  3. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.ac.jp; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu, E-mail: katsuma@ss.ab.a.u-tokyo.ac.jp

    2016-11-15

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  4. UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

    Science.gov (United States)

    Lamore, Sarah D.; Wondrak, Georg T.

    2013-01-01

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

  5. Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

    Science.gov (United States)

    Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

    2017-08-01

    Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  6. Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

    Science.gov (United States)

    Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

    2017-12-01

    Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

    Science.gov (United States)

    Shen, Jian-Dong; Cai, Qiu-Feng; Yan, Long-Jie; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Ke, Caihuan; Cao, Min-Jie

    2015-12-01

    Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Cathepsin D, a Marker for the Metastatic Potential of Breast Cancer, May Regulate the Mitogenic Activity of Fibroblast Growth Factor 1

    National Research Council Canada - National Science Library

    Grieb, Teri

    1998-01-01

    .... Over the years, the data substantiating such a role for cathepsin D has been quite conflicting However, there is strong evidence that cathepsin D plays a role in the degradation of the extracellular matrix (ECM...

  9. Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: Mutations in the 25K FP gene may be caused by DNA replication errors

    Science.gov (United States)

    David S. Bischoff; James M. Slavicek

    1997-01-01

    We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J.M. Slavicek, N. Hayes-Plazolles, and M.E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants...

  10. Autographa californica Multicapsid Nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH

    NARCIS (Netherlands)

    Dong, S.; Wang, M.; Qiu, Z.; Deng, F.; Vlak, J.M.; Hu, Z.H.; Wang, H.L.

    2010-01-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue

  11. Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs are functionally analogous to AcMNPV GP64

    NARCIS (Netherlands)

    Lung, O.; Westenberg, M.; Vlak, J.M.; Zuidema, D.; Blissard, G.W.

    2002-01-01

    GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral

  12. Prevalence and clinical significance of cathepsin G antibodies in systemic sclerosis

    Directory of Open Access Journals (Sweden)

    M. Favaro

    2011-09-01

    Full Text Available Objectives: To evaluate the prevalence and clinical significance of cathepsin G antibodies in patients affected with systemic sclerosis (SSc, scleroderma. Methods: 115 patients affected by SSc, 55 (47,8% with diffuse scleroderma (dSSc and 60 (52,2% with limited scleroderma (lSSc, were tested for cathepsin G antibodies by ELISA method. Moreover these sera were evaluated by indirect immunofluorescence (IIF on ethanol and formalin fixed human neutrophils. Results: By means of the ELISA method 16 (13,9% patients were found to be sera positive for anti-cathepsin G, 2 (12.5% of which showed a perinuclear fluorescence pattern (P-ANCA and 4 (25% an atypical ANCA staining, while 10 (62,5% were negative on IIF. The IIF on scleroderma sera revealed 5 (4,3% P-ANCA and 18 (15,7% atypical ANCA patterns. The anti-cathepsin G antibodies significantly prevailed in scleroderma sera (p=0.02 when their frequency was compared with that of healthy controls; while they were not significantly associated to any clinical or serological features of SSc patients. Conclusions: The anti-cathepsin G antibodies were significantly frequent in scleroderma sera; however, no clinical correlations were found. Thus, the significance of their presence in SSc still needs to be clarified.

  13. Expression, purification and auto-activation of cathepsin E from insect cells.

    Science.gov (United States)

    Železnik, Tajana Z; Puizdar, Vida; Dolenc, Iztok

    2015-01-01

    Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.

  14. Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

    Science.gov (United States)

    Liang, Pei; He, Lei; Xu, Yanquan; Chen, Xueqing; Huang, Yan; Ren, Mengyu; Liang, Chi; Li, Xuerong; Xu, Jin; Lu, Gang; Yu, Xinbing

    2014-10-01

    Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

  15. Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

    Directory of Open Access Journals (Sweden)

    Andreas Koeberle

    2018-02-01

    Full Text Available Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO pathway. Boswellic acids (BAs are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

  16. Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G.

    Science.gov (United States)

    Koeberle, Andreas; Henkel, Arne; Verhoff, Moritz; Tausch, Lars; König, Stefanie; Fischer, Dagmar; Kather, Nicole; Seitz, Stefanie; Paul, Michael; Jauch, Johann; Werz, Oliver

    2018-02-24

    Age-related diseases, such as osteoarthritis, Alzheimer's disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

  17. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    International Nuclear Information System (INIS)

    Inami, Yoshihiro; Yamashina, Shunhei; Izumi, Kousuke; Ueno, Takashi; Tanida, Isei; Ikejima, Kenichi; Watanabe, Sumio

    2011-01-01

    Highlights: → Acidification of autophagosome was blunted in steatotic hepatocytes. → Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. → Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. → Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  18. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    Energy Technology Data Exchange (ETDEWEB)

    Inami, Yoshihiro [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Tanida, Isei [Department of Biochemistry and Cell Biology, Laboratory of Biomembranes, National Institute of Infectious Disease, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640 (Japan); Ikejima, Kenichi; Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  19. Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

    Science.gov (United States)

    Tsujimura, Atsushi; Taguchi, Katsutoshi; Watanabe, Yoshihisa; Tatebe, Harutsugu; Tokuda, Takahiko; Mizuno, Toshiki; Tanaka, Masaki

    2015-01-01

    The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils. Copyright © 2015. Published by Elsevier Inc.

  20. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    Directory of Open Access Journals (Sweden)

    Altmeyer Peter

    2002-08-01

    Full Text Available Abstract Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2 has achieved great value within the treatment of severe atopic dermatitis (AD. The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with AD, biopsy specimens from 15 patients before and after treatment were analyzed immunohistochemically for proteolytic activation. Results Compared to lesional skin of patients with AD before UVA1 irradiation, the number of cells positive for cathepsin G within the dermal infiltrate decreased significantly after treatment. The decrease of cathepsin G+ cells was closely linked to a substantial clinical improvement in skin condition. Conclusions In summary, our findings demonstrated that medium-dose UVA1 irradiation leads to a modulation of the expression of cathepsin G in the dermal inflammatory infiltrate in patients with severe AD. Cathepsin G may attack laminin, proteoglycans, collagen I and insoluble fibronectin, to provoke proinflammatory events, to degrade the basement membrane, to destroy the tissue inhibitor of metalloproteinases and to increase the endothelial permeability. Therefore, its down-regulation by UVA1 phototherapy may induce the reduction of skin inflammation as well as improvement of the skin condition.

  1. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    Science.gov (United States)

    Breuckmann, Frank; von Kobyletzki, Gregor; Avermaete, Annelies; Kreuter, Alexander; Altmeyer, Peter; Gambichler, Thilo

    2002-01-01

    Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great value within the treatment of severe atopic dermatitis (AD). The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with AD, biopsy specimens from 15 patients before and after treatment were analyzed immunohistochemically for proteolytic activation. Results Compared to lesional skin of patients with AD before UVA1 irradiation, the number of cells positive for cathepsin G within the dermal infiltrate decreased significantly after treatment. The decrease of cathepsin G+ cells was closely linked to a substantial clinical improvement in skin condition. Conclusions In summary, our findings demonstrated that medium-dose UVA1 irradiation leads to a modulation of the expression of cathepsin G in the dermal inflammatory infiltrate in patients with severe AD. Cathepsin G may attack laminin, proteoglycans, collagen I and insoluble fibronectin, to provoke proinflammatory events, to degrade the basement membrane, to destroy the tissue inhibitor of metalloproteinases and to increase the endothelial permeability. Therefore, its down-regulation by UVA1 phototherapy may induce the reduction of skin inflammation as well as improvement of the skin condition. PMID:12204095

  2. Identification and characterization of the novel reversible and selective cathepsin X inhibitors.

    Science.gov (United States)

    Fonović, Urša Pečar; Mitrović, Ana; Knez, Damijan; Jakoš, Tanja; Pišlar, Anja; Brus, Boris; Doljak, Bojan; Stojan, Jure; Žakelj, Simon; Trontelj, Jurij; Gobec, Stanislav; Kos, Janko

    2017-09-13

    Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R 1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (K i  = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

  3. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  4. Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells

    NARCIS (Netherlands)

    Rijnboutt, S.; Kal, A. J.; Geuze, H. J.; Aerts, H.; Strous, G. J.

    1991-01-01

    We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically

  5. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lyupina, Yulia V.; Abaturova, Svetlana B.; Erokhov, Pavel A. [N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334 (Russian Federation); Orlova, Olga V.; Beljelarskaya, Svetlana N. [V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119334 (Russian Federation); Mikhailov, Victor S., E-mail: mikhailov48@mail.ru [N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilova Str., Moscow 119334 (Russian Federation)

    2013-02-05

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24-48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  6. Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

    International Nuclear Information System (INIS)

    Lyupina, Yulia V.; Abaturova, Svetlana B.; Erokhov, Pavel A.; Orlova, Olga V.; Beljelarskaya, Svetlana N.; Mikhailov, Victor S.

    2013-01-01

    Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24–48 hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

  7. Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

    Science.gov (United States)

    Zhang, Min-Juan; Tian, Cai-Hong; Fan, Xiao-Ying; Lou, Yi-Han; Cheng, Ruo-Lin; Zhang, Chuan-Xi

    2012-07-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

  8. Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Cheng, Xiao-Wen; Carner, Gerald R; Lange, Martin; Jehle, Johannes A; Arif, Basil M

    2005-02-01

    A multicapsid nucleopolyhedrovirus (ThorMNPV) that was co-isolated with a single nucleocapid ThorSNPV from mixed infected larvae of Thysanoplusia orichalcea L. (Lepidoptea: Noctuidae) is characterized. Scanning electron microscopy of ThorMNPV showed a dodecahedral-shaped occlusion body (OB). The occluded virions contained one to as many as eight nucleocapsids/virion. Virion band profiles in gradient centrifugation were consistent in at least 10 rounds of centrifugation from different virion sample preparations. The ThorMNPV had high virulence to third instar Trichoplusia ni and Pseudoplusia includens with LD50 values of 17 and 242OBs per larva, respectively. However, ThorMNPV did not cause mortality in Spodoptera exigua, Spodoptera frugiperda, Spodoptera eridania, Anticarsia gemmatalis, and Helicoverpa zea. ThorMNPV replicates in cells of various tissues such as the fat body and tracheal epithelium cells. T. ni High 5 cells were permissive to ThorMNPV in terms of infection and viral DNA transfection, but SF-21 was less permissive and the infection process was slower. Production of OBs by ThorMNPV in the nuclei of SF-21 was not well pronounced. The genome size of ThorMNPV was estimated to be 136 kb. The polyhedrin gene open reading frame (ORF) was cloned and completely sequenced. The promoter sequence is identical to that of Autographa californica MNPV. Phylogenetic analyses using partial sequences of the polh, lef-8, and lef-9 revealed that ThorMNPV is a member of the Group I NPVs and is related but distinct from the AcMNPV/Rachiplusia ou NPV/Bombyx mori NPV cluster.

  9. Characterization of a single-nucleocapsid nucleopolyhedrovirus of Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia.

    Science.gov (United States)

    Cheng, X W; Carner, G R

    2000-05-01

    A single-nucleocapsid nucleopolyhedrovirus (NPV) isolated from Thysanoplusia orichalcea L. (Lepidoptera:Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1. 22 microm (range = 0.803-1.931 microm). The length of the virion with an envelope averaged 0.29 and 0.23 microm without an envelope. ThorNPV was propagated in Pseudoplusia includens (Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV produced in T. orichalcea and P. includens. Polyhedrin amino acid sequence analysis revealed that ThorNPV belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV against third instar P. includens was low, with a LD(50) value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions embedded, which might explain the low virulence against P. includens. Differences in virion occlusion rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD(50) value to P. includens against Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens, P. includens was the only species infected. The virus infected primarily the fat body, tracheal epithelium, and hypodermis. The genomic size of the ThorNPV is 135 kb. Copyright 2000 Academic Press.

  10. Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva

    Directory of Open Access Journals (Sweden)

    Wang Yong

    2009-02-01

    Full Text Available Abstract Background Baculoviruses are well known for their potential as biological agents for controlling agricultural and forest pests. They are also widely used as expression vectors in molecular cloning studies. The genome sequences of 48 baculoviruses are currently available in NCBI databases. As the number of sequenced viral genomes increases, it is important for the authors to present sufficiently detailed analyses and annotations to advance understanding of them. In this study, the complete genome of Clanis bilineata nucleopolyhedrovirus (ClbiNPV has been sequenced and analyzed in order to understand this virus better. Results The genome of ClbiNPV contains 135,454 base pairs (bp with a G+C content of 37%, and 139 putative open reading frames (ORFs of at least 150 nucleotides. One hundred and twenty-six of these ORFs have homologues with other baculovirus genes while the other 13 are unique to ClbiNPV. The 30 baculovirus core genes are all present in ClbiNPV. Phylogenetic analysis based on the combined pif-2 and lef-8 sequences places ClbiNPV in the Group II Alphabaculoviruses. This result is consistent with the absence of gp64 from the ClbiNPV genome and the presence instead of a fusion protein gene, characteristic of Group II. Blast searches revealed that ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion disrupts the sequence into two small photolyase ORFs, designated Clbiphr-1 and Clbiphr-2, which correspond to the CPD-DNA photolyase and FAD-binding domains of photolyases, respectively. Conclusion ClbiNPV belongs to the Group II Alphabaculoviruses and is most closely related to OrleNPV, LdMNPV, TnSNPV, EcobNPV and ChchNPV. It contains a variant DNA photolyase gene, which only exists in ChchNPV, TnSNPV and SpltGV among the baculoviruses.

  11. Effects of cetyltriethylammonium bromide on the replication of Bombyx mori nucleopolyhedrovirus.

    Science.gov (United States)

    Zhou, Yajing; Zhang, Zhifang; He, Jialu; Zhang, Yuanxing

    2002-05-01

    An experimental study was undertaken to quantify the effects of cetyltriethylammonium bromide (CTAB) on the replication of Bombyx mori nucleopolyhedrovirus (BmNPV) and the transcriptionalactivity of BmNPV ie-1 promoter. The results demonstrated that the budded virus (BV) titer rose about 3.7-fold by adding CTAB to the culture media up to 0.1 mu g ml(-1) in infected Bm-N cells with a wild-type BmNPV. The transient expression level of luciferase driven by BmNPV ie-1 promoter was enhanced by more than 3-fold in the presence of 0.1 mu g ml(-1) of CTAB in uninfected insect cells via a transient expression system. Contrary to the rise in BV titer, the polyhedra inside the nucleus of infected cells dropped linearly from 4.0 x 10(6) ml(-1) down to 2.1 x 10(6) ml(-1) with in a range of CTAB concentrations from 0 to 0.25 mu g ml(-1). The same trend in expression level of beta -galactosidase or phytase was given when the Bm-N cells or fifth-instar silkworm larvae infected with a recombinant BmNPV containing the beta -galactosidase or phytase reporter gene driven by the polyhedrin promoter. We deduced that CTAB appeared to affect the virus bi-phasic life cycle stages and production pathways, resulting in an enhancement in BV production and a suppression of occluded virus (OV) production and expression of foreign genes controlled by the polyhedrin promoter.

  12. Functional characterization of Bombyx mori nucleopolyhedrovirus mutant lacking late expression factor 9.

    Science.gov (United States)

    Zhang, Y; Shi, Y; Yu, H; Li, J; Quan, Y; Shu, T; Nie, Z; Zhang, Y; Yu, W

    Baculoviridae is a family of invertebrate viruses with large double-stranded DNA genomes. Proteins encoded by some late expression factor (lef ) genes are involved in the regulation of viral gene expression. Lef-9 is one of four transcription-specific Lefs, which are components of the virus-encoded RNA polymerase, and can initiate and transcribe late and very late genes. As a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus (BmNPV), Lef-9 may be involved in the regulation of viral propagation. However, the underlying mechanism remains unclear. To determine the role of lef-9 in baculovirus infection, lef-9-knockout virus (lef-9-KO-Bacmid virus) was constructed using the Red recombination system, and the Bac-to-Bac system was used to prepare lef-9-repaired virus (lef-9-Re-Bacmid virus). The lef-9-KO virus did not produce infectious viruses or show infection activity, while the lef-9-repaired virus recovered both. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the transcription levels in wild-type-Bacmid, lef-9-KO-Bacmid, and lef-9-Re-Bacmid viruses showed that the lef-9-KO bacmid had little effect on viral genome replication. However, the transcription levels of the early and late viral genes, lef-3, ie-1, vp39, and p10, were significantly lower in BmN cells transfected with lef-9-KO-Bacmids than in the controls. Electron microscopy showed no visible enveloped virions in cells transfected with lef-9-KO-Bacmids, while many mature virions in cells transfected with lef-9-Re-Bacmid and wt-Bacmid were present. Thus, lef-9 was not essential for viral genome replication, but significantly affected viral gene transcription and expression in all periods of cell life cycle.

  13. Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

    Science.gov (United States)

    Hu, Xiaolong; Zhu, Min; Liang, Zi; Kumar, Dhiraj; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2017-04-01

    The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

  14. V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus.

    Directory of Open Access Journals (Sweden)

    Peng Lü

    Full Text Available Silkworms are usually susceptible to the infection of Bombyx mori (B. mori nucleopolyhedrovirus (BmNPV, which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells of BmNPV-resistant strains (NB and BC8, the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi, the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

  15. Kinetic analysis of in vitro production of wild-type Spodoptera frugiperda nucleopolyhedrovirus

    Directory of Open Access Journals (Sweden)

    Andréa Farias de Almeida

    2010-04-01

    Full Text Available In this study, the kinetic behavior of Sf9 and Sf21 cells used in the production of a baculovirus biopesticide to control the pest of corn Spodoptera frugiperda was analyzed. Kinetic variables such as maximum specific growth rate, cell productivity, mean rate of infection, as well as the mean rate of occlusion body production were determined during the infection of these cell-lines with the extracellular virus of the S. frugiperda nucleopolyhedrovirus (SfMNPV. The Sf9 cell-line resulted in better viral production results (5.0 x 10(8 OB/mL than the Sf21 cell-line (2.5 x 10(8 OB/mL.Neste trabalho, analisou-se o comportamento cinético das células Sf9 e Sf21 utilizadas na produção de biopesticida para o controle de Spodoptera frugiperda. Variáveis cinéticas, como velocidade específica máxima de crescimento, produtividade em células, velocidade média de infecção e a velocidade média de produção de OB foram determinadas durante a infecção destas linhagens com o vírus extracelular do nucleopoliedrovirus de S. frugiperda. A linhagem Sf9 resultou em melhores resultados de produção do baculovírus (5 x 10(8 OB/mL, quando comparada à linhagem Sf21 (2,5 x 10(8 OB/mL e outras linhagens da literatura.

  16. Nucleopolyhedrovirus detection and distribution in terrestrial, freshwater, and marine habitats of Appledore Island, Gulf of Maine.

    Science.gov (United States)

    Hewson, Ian; Brown, Julia M; Gitlin, Shari A; Doud, Devin F

    2011-07-01

    Viruses in aquatic ecosystems comprise those produced by both autochthonous and allochthonous host taxa. However, there is little information on the diversity and abundance of viruses of allochthonous origin, particularly from non-anthropogenic sources, in freshwater and marine ecosystems. We investigated the presence of nucleopolyhedroviruses (NPV) (Baculovirus), which commonly infect terrestrial lepidopteran taxa, across the landscape of Appledore Island, Gulf of Maine. PCR and qPCR primers were developed around a 294-bp fragment of the polyhedrin (polH) gene, which is the major constituent protein of NPV multivirion polyhedral occlusion bodies. polH was successfully amplified from several aquatic habitats, and recovered polH sequences were most similar to known lepidopteran NPV. Using quantitative PCR designed around a cluster of detected sequences, we detected polH in Appledore Island soils, supratidal freshwater ponds, nearshore sediments, near- and offshore plankton, and in floatsam. This diverse set of locations suggests that NPVs are widely dispersed along the terrestrial--marine continuum and that free polyhedra may be washed into ponds and eventually to sea. The putative hosts of detected NPVs were webworms (Hyphantria sp.) which form dense nests in late summer on the dominant Appledore Island vegetation (Prunus virginiana). Our data indicate that viruses of terrestrial origin (i.e., allochthonous viruses) may be dispersed widely in coastal marine habitats. The dispersal of NPV polH and detection within offshore net plankton (>64 μm) demonstrates that terrestrial viruses may interact with larger particles and plankton of coastal marine ecosystem, which further suggests that viral genomic information may be transported between biomes.

  17. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

    Science.gov (United States)

    Shoji, Atsushi; Suenaga, Yumiko; Hosaka, Atsushi; Ishida, Yuuki; Yanagida, Akio; Sugawara, Masao

    2017-10-25

    We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

    DEFF Research Database (Denmark)

    Delaissé, Jean-Marie; Andersen, Thomas L; Engsig, Michael T

    2003-01-01

    The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute...... significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone...... in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations...

  19. Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

    Directory of Open Access Journals (Sweden)

    Porntip Pinlaor

    Full Text Available The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and

  20. Cathepsin B & L are not required for ebola virus replication.

    Science.gov (United States)

    Marzi, Andrea; Reinheckel, Thomas; Feldmann, Heinz

    2012-01-01

    Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

  1. Cathepsin B & L are not required for ebola virus replication.

    Directory of Open Access Journals (Sweden)

    Andrea Marzi

    Full Text Available Ebola virus (EBOV, family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV. EBOV encodes one viral surface glycoprotein (GP, which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL, which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control, catB(-/- and catL(-/- mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

  2. Cathepsin K expression and activity in canine osteosarcoma.

    Science.gov (United States)

    Schmit, J M; Pondenis, H C; Barger, A M; Borst, L B; Garrett, L D; Wypij, J M; Neumann, Z L; Fan, T M

    2012-01-01

    Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations. Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption. Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs. In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents. Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFβ1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03. Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis. Copyright © 2011 by the American College of Veterinary Internal Medicine.

  3. Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

    Science.gov (United States)

    Gatto, Barbara; Vianini, Elena; Lucatello, Lorena; Sissi, Claudia; Moltrasio, Danilo; Pescador, Rodolfo; Porta, Roberto; Palumbo, Manlio

    2008-01-01

    Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions. PMID:19325843

  4. Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

    Directory of Open Access Journals (Sweden)

    Manlio Palumbo

    2008-06-01

    Full Text Available Cathepsin G (CatG is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions.

  5. Activation of lysosomal cathepsins in pregnant bovine leukocytes.

    Science.gov (United States)

    Talukder, Md Abdus Shabur; Balboula, Ahmed Zaky; Shirozu, Takahiro; Kim, Sung Woo; Kunii, Hiroki; Suzuki, Toshiyuki; Ito, Tsukino; Kimura, Koji; Takahashi, Masashi

    2018-06-01

    In ruminants, interferon-tau (IFNT) - mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy. © 2018 Society for Reproduction and Fertility.

  6. Cathepsin K expression is increased in oral lichen planus.

    Science.gov (United States)

    Siponen, Maria; Bitu, Carolina Cavalcante; Al-Samadi, Ahmed; Nieminen, Pentti; Salo, Tuula

    2016-11-01

    Oral lichen planus (OLP) is an idiopathic T-cell-mediated mucosal inflammatory disease. Cathepsin K (Cat K) is one of the lysosomal cysteine proteases. It is involved in many pathological conditions, including osteoporosis and cancer. The expression and role of Cat K in OLP are unknown. Twenty-five oral mucosal specimens diagnosed histopathologically as OLP and fourteen healthy controls (HC) were used to study the immunohistochemical (IHC) expression of Cat K. Colocalization of Cat K with CD1a, Melan-A, CD68, CD45, mast cell tryptase (MCT), and Toll-like receptors (TLRs) 4 and 9 were studied using double IHC and/or immunofluorescence (IF) staining. Expression of Cat K was also evaluated in OLP tissue samples before and after topical tacrolimus treatment. Cat K was expressed in a higher percentage of cells in the epithelial zone, and the staining intensity was stronger in the stroma in OLP compared to controls (P < 0.001). In OLP, Cat K was present mostly in melanocytes and macrophages and sporadically in basal keratinocytes, endothelial cells, and extracellularly. Cat K was found also in some fibroblasts in HC and OLP samples. Coexpression of Cat K and TLRs 4 and 9 was seen in some dendritic cells (presumably melanocytes) and macrophages. In OLP, tacrolimus treatment reduced the expression of Cat K in the epithelium but increased it in the stroma. These results suggest that Cat K is involved in the pathogenesis of OLP. Cat K possibly takes part in the modulation of matrix molecules and cellular receptors. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus).

    Science.gov (United States)

    Liu, Yu-Xin; Zhou, Da-Yong; Ma, Dong-Dong; Liu, Yan-Fei; Li, Dong-Mei; Dong, Xiu-Ping; Tan, Ming-Qian; Du, Ming; Zhu, Bei-Wei

    2016-12-01

    The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Cathepsin Gene Family Reveals Transcriptome Patterns Related to the Infective Stages of the Salmon Louse Caligus rogercresseyi.

    Directory of Open Access Journals (Sweden)

    Waleska Maldonado-Aguayo

    Full Text Available Cathepsins are proteases involved in the ability of parasites to overcome and/or modulate host defenses so as to complete their own lifecycle. However, the mechanisms underlying this ability of cathepsins are still poorly understood. One excellent model for identifying and exploring the molecular functions of cathepsins is the marine ectoparasitic copepod Caligus rogercresseyi that currently affects the Chilean salmon industry. Using high-throughput transcriptome sequencing, 56 cathepsin-like sequences were found distributed in five cysteine protease groups (B, F, L, Z, and S as well as in an aspartic protease group (D. Ontogenic transcriptome analysis evidenced that L cathepsins were the most abundant during the lifecycle, while cathepsins B and K were mostly expressed in the larval stages and adult females, thus suggesting participation in the molting processes and embryonic development, respectively. Interestingly, a variety of cathepsins from groups Z, L, D, B, K, and S were upregulated in the infective stage of copepodid, corroborating the complexity of the processes involved in the parasitic success of this copepod. Putative functional roles of cathepsins were conjectured based on the differential expressions found and on roles previously described in other phylogenetically related species. Moreover, 140 single nucleotide polymorphisms (SNP were identified in transcripts annotated for cysteine and aspartic proteases located into untranslated regions, or the coding region. This study reports for the first time the presence of cathepsin-like genes and differential expressions throughout a copepod lifecycle. The identification of cathepsins together with functional validations represents a valuable strategy for pinpointing target molecules that could be used in the development of new delousing drugs or vaccines against C. rogercresseyi.

  9. Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

    Science.gov (United States)

    Gikanga, Benson; Adeniji, Nia S; Patapoff, Thomas W; Chih, Hung-Wei; Yi, Li

    2016-04-20

    Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different

  10. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion.

    Science.gov (United States)

    de Mingo Pulido, Álvaro; de Gregorio, Estefanía; Chandra, Shilpi; Colell, Anna; Morales, Albert; Kronenberg, Mitchell; Marí, Montserrat

    2018-01-01

    Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs), probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro .

  11. Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein

    Science.gov (United States)

    Puizdar, Vida; Zajc, Tajana; Žerovnik, Eva; Renko, Miha; Pieper, Ursula; Eswar, Narayanan; Šali, Andrej; Dolenc, Iztok; Turk, Vito

    2014-01-01

    Cathepsin E splice variant 2 appears in a number of gastric carcinoma. Here, we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variant 1 and variant 2 of cathepsins E, with propeptide and without it, in Echerichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by Atomic Force Microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS), and used to rationalize its conformational properties and loss of activity. PMID:22718633

  12. Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori

    International Nuclear Information System (INIS)

    Skvarc, Miha; Stubljar, David; Kopitar, Andreja Natasa; Jeverica, Samo; Tepes, Bojan; Kos, Janko; Ihan, Alojz

    2013-01-01

    The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed

  13. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    Directory of Open Access Journals (Sweden)

    Álvaro de Mingo Pulido

    2018-02-01

    Full Text Available Natural killer T (NKT cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ, and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB and cathepsin S (CTSS, regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs, probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro.

  14. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Roč. 90, č. 3 (2008), s. 349-357 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

  15. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Goyal, Shruti; Amar, Saroj Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Dubey, Divya; Pal, Manish Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Singh, Jyoti [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Verma, Ankit; Kushwaha, Hari Narayan [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Ray, Ratan Singh, E-mail: ratanray.2011@rediffmail.com [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India)

    2015-12-30

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  16. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    International Nuclear Information System (INIS)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-01-01

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  17. Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth.

    Directory of Open Access Journals (Sweden)

    Elias Gounaris

    2008-08-01

    Full Text Available It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4 of the probe signal was localized in CD11b(+Gr1(+ myeloid derived suppressor cells (MDSC and CD11b(+F4/80(+ macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.

  18. Prognostic and predictive value of cathepsin X in serum from colorectal cancer patients

    DEFF Research Database (Denmark)

    Vižin, Tjaša; Christensen, Ib Jarle; Wilhelmsen, Michael

    2014-01-01

    , but for patients in stages I-III with local resectable disease. The significant association of cathepsin X with survival in a group of patients who received no chemotherapy and the absence of this association in the group who received chemotherapy, suggest the possible predictive value for response to chemotherapy...

  19. A novel nonsense mutation in cathepsin C gene in an Egyptian ...

    African Journals Online (AJOL)

    Background: Cathepsin C gene (CTSC) (MIM#602365) is a lysosomal cysteine proteinase coding gene which encodes for CTSC protein that plays a major role in the activation of granule serine proteases, particularly leukocyte elastase and granzymes A and B. This activity was proposed to play a role in epithelial ...

  20. Oestrogen regulates the expression of cathepsin E-A-like gene ...

    Indian Academy of Sciences (India)

    Hang Zheng

    2018-02-28

    Feb 28, 2018 ... 1College of Animal Science and Veterinary Medicine, Henan Agricultural .... evaluated the expression regulation mechanism of the gene ... C with ad libitum water and food. ... embryonic liver following the method previously described .... Cloning and sequence analysis of chicken cathepsin E-A-like gene.

  1. Cathepsin L is crucial for the development of early experimental diabetic nephropathy

    NARCIS (Netherlands)

    Garsen, M.; Rops, A.; Dijkman, H.B.; Willemsen, B.K.; Kuppevelt, T.H. van; Russel, F.G.M.; Rabelink, T.J.; Berden, J.H.; Reinheckel, T.; Vlag, J. van der

    2016-01-01

    Proteinuria is one of the first clinical signs of diabetic nephropathy and an independent predictor for the progression to renal failure. Cathepsin L, a lysosomal cysteine protease, can be involved in the development of proteinuria by degradation of proteins that are important for normal podocyte

  2. Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets

    Czech Academy of Sciences Publication Activity Database

    Sojka, D.; Hartmann, D.; Bartošová-Sojková, P.; Dvořák, Jan

    2016-01-01

    Roč. 32, č. 9 (2016), s. 708-723 ISSN 1471-4922 R&D Projects: GA ČR GA13-11043S; GA ČR(CZ) GAP302/11/1481 Institutional support: RVO:61388963 Keywords : aspartic peptidases * cathepsin D * hemoglobinolysis * parasites * vectors Subject RIV: CE - Biochemistry Impact factor: 6.333, year: 2016

  3. Activation route of the Schistosoma mansoni cathepsin B1 drug target

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Horn, Martin; Řezáčová, Pavlína; Marešová, Lucie; Fajtová, Pavla; Brynda, Jiří; Vondrášek, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 39 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : Schistosoma mansoni * cathepsin B1 * sulfated polysaccharides Subject RIV: CE - Biochemistry

  4. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    Energy Technology Data Exchange (ETDEWEB)

    Schreuder, Herman A., E-mail: herman.schreuder@sanofi.com; Liesum, Alexander, E-mail: alexander.liesum@sanofi.com; Kroll, Katja, E-mail: katja.kroll@sanofi.com; Böhnisch, Britta, E-mail: britta.boehnisch@sanofi.com; Buning, Christian, E-mail: christian.buning@sanofi.com; Ruf, Sven, E-mail: sven.ruf@sanofi.com; Sadowski, Thorsten, E-mail: thorsten.sadowski@sanofi.com

    2014-03-07

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  5. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

    International Nuclear Information System (INIS)

    Kang, Won Kyung; Kurihara, Masaaki; Matsumoto, Shogo

    2006-01-01

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

  6. Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer.

    Science.gov (United States)

    Chen, Binbin; Platt, Manu O

    2011-07-14

    Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties. Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity. Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients. To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.

  7. Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

    Science.gov (United States)

    Wilder, Catera L; Park, Keon-Young; Keegan, Philip M; Platt, Manu O

    2011-12-01

    Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Salivary Tick Cystatin OmC2 Targets Lysosomal Cathepsins S and C in Human Dendritic Cells

    Czech Academy of Sciences Publication Activity Database

    Zavasnik-Bergant, T.; Vidmar, R.; Sekirnik, A.; Fonovic, M.; Salát, Jiří; Grunclová, Lenka; Kopáček, Petr; Turk, B.

    2017-01-01

    Roč. 7, JUN 30 (2017), č. článku 288. ISSN 2235-2988 R&D Projects: GA ČR GA13-11043S Institutional support: RVO:60077344 Keywords : cystatin OmC2 * tick saliva * cathepsin S * cathepsin C * lysosomal proteases * dpp1 * dipeptidyl peptidase 1 * dendritic cells Subject RIV: EC - Immunology OBOR OECD: Immunology Impact factor: 4.300, year: 2016

  9. Study of Low-grade Chronic Inflammatory Markers in Men with Central Obesity: Cathepsin S was Correlated with Waist Circumference

    Directory of Open Access Journals (Sweden)

    Adriana Todingrante

    2013-08-01

    Full Text Available BACKGROUND: There is a prevalence increase of overweight and obesity in Indonesia. Central obesity can lead a variety of chronic diseases through the inflammatory process. There are some markers for low-grade chronic inflammatory, such as cathepsin S, high sensitivity C-reactive protein (hs-CRP, interleukin-1- beta (IL-1β. To our current interest that central obesity can lead to various chronic diseases through the inflammatory process, we conducted a study to investigate correlation of Cathepsin S, hs-CRP, IL-1β in men with central obesity. METHODS: A cross-sectional study was conducted. Seventy-eight selected subjects were examined to collect anthropometric data and prepared for sample collection. Collected samples were processed for the following biochemical analyses: fasting glucose, high density lipoprotein (HDL-cholesterol, triglyceride, serum glutamic oxaloacetic transaminase (SGOT, serum glutamate pyruvate transaminase (SGPT, cathepsin S, hs-CRP, and IL-1β. Data distribution and variable correlation were then statistically analyzed. RESULTS: There were significant correlations between waist circumference (WC and cathepsin S (p=0.030; r=0.214, hs-CRP and cathepsin S (p=0.007; r=0.276, triglyceride and IL-1β (p=0.019; r=-0.235, WC and systolic blood pressure (SBP (p=0.003; r=-0.312, WC and fasting glucose (p=0.000; r=0.380, WC and body mass index (BMI (p=0.000; r=0.708. CONCLUSIONS: Our study showed that cathepsin S was correlated with central obesity, suggesting that cathepsin S could be a potential inflammatory marker in central obesity in the future. KEYWORDS: obesity, inflammation, hs-CRP, cathepsin S, IL-1β, waist circumference.

  10. Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period.

    Science.gov (United States)

    García, Víctor; Kohen, Paulina; Maldonado, Carola; Sierralta, Walter; Muñoz, Alex; Villarroel, Claudio; Strauss, Jerome F; Devoto, Luigi

    2012-03-01

    To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. Experimental study. University research unit. Twenty-five women of reproductive age. Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation. Copyright © 2012 American Society for Reproductive Medicine. All rights reserved.

  11. Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

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    Mark W Robinson

    Full Text Available BACKGROUND: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3, liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains. Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. CONCLUSIONS/SIGNIFICANCE: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.

  12. Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Skjøt-Arkil, Helene; Register, Thomas C

    2010-01-01

    BACKGROUND: Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular...... matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes. METHODS: We 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries......-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in women...

  13. Effects of Chilling and Partial Freezing on Rigor Mortis Changes of Bighead Carp (Aristichthys nobilis) Fillets: Cathepsin Activity, Protein Degradation and Microstructure of Myofibrils.

    Science.gov (United States)

    Lu, Han; Liu, Xiaochang; Zhang, Yuemei; Wang, Hang; Luo, Yongkang

    2015-12-01

    To investigate the effects of chilling and partial freezing on rigor mortis changes in bighead carp (Aristichthys nobilis), pH, cathepsin B, cathepsin B+L activities, SDS-PAGE of sarcoplasmic and myofibrillar proteins, texture, and changes in microstructure of fillets at 4 °C and -3 °C were determined at 0, 2, 4, 8, 12, 24, 48, and 72 h after slaughter. The results indicated that pH of fillets (6.50 to 6.80) was appropriate for cathepsin function during the rigor mortis. For fillets that were chilled and partially frozen, the cathepsin activity in lysosome increased consistently during the first 12 h, followed by a decrease from the 12 to 24 h, which paralleled an increase in activity in heavy mitochondria, myofibrils and sarcoplasm. There was no significant difference in cathepsin activity in lysosomes between fillets at 4 °C and -3 °C (P > 0.05). Partially frozen fillets had greater cathepsin activity in heavy mitochondria than chilled samples from the 48 to 72 h. In addition, partially frozen fillets showed higher cathepsin activity in sarcoplasm and lower cathepsin activity in myofibrils compared with chilled fillets. Correspondingly, we observed degradation of α-actinin (105 kDa) by cathepsin L in chilled fillets and degradation of creatine kinase (41 kDa) by cathepsin B in partially frozen fillets during the rigor mortis. The decline of hardness for both fillets might be attributed to the accumulation of cathepsin in myofibrils from the 8 to 24 h. The lower cathepsin activity in myofibrils for fillets that were partially frozen might induce a more intact cytoskeletal structure than fillets that were chilled. © 2015 Institute of Food Technologists®

  14. Atick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

    Czech Academy of Sciences Publication Activity Database

    Chmelař, Jindřich; Oliveira, C. J.; Řezáčová, Pavlína; Francischetti, I.M.B.; Kovářová, Zuzana; Pejler, G.; Kopáček, Petr; Ribeiro, J.M.C.; Mareš, Michael; Kopecký, Jan; Kotsyfakis, Michalis

    2011-01-01

    Roč. 117, č. 2 (2011), s. 736-744 ISSN 0006-4971 R&D Projects: GA AV ČR IAA600960811; GA MŠk(CZ) LC06009; GA ČR(CZ) GAP207/10/2183 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : parasite serpin * IRS-2 * tick * Ixodes ricinus * platelet aggregation * inflammation * cathepsin G * chymase Subject RIV: EC - Immunology Impact factor: 9.898, year: 2011

  15. An Ectosteric Inhibitor of Cathepsin K Inhibits Bone Resorption in Ovariectomized Mice

    DEFF Research Database (Denmark)

    Panwar, Preety; Xue, Liming; Søe, Kent

    2017-01-01

    The potent cathepsin K (CatK) inhibitor, Tanshinone IIA sulfonic sodium (T06), was tested for its in vitro and in vivo antiresorptive activities. T06 binds in an ectosteric site of CatK remote from its active site and selectively inhibits collagen degradation with an IC50 value of 2.7±0.2μM (CatK...

  16. Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Řezáčová, Pavlína; Lepšík, Martin; Horn, Martin; Váchová, Jana; Fanfrlík, Jindřich; Brynda, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2011-01-01

    Roč. 286, č. 41 (2011), s. 35770-35881 ISSN 0021-9258 R&D Projects: GA ČR GA203/09/1585; GA ČR GAP208/11/0295; GA MŠk OC09007 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : cathepsin B * schistosoma * crystal structure Subject RIV: CC - Organic Chemistry Impact factor: 4.773, year: 2011

  17. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

  18. The role of cathepsin E in the antigen processing and presentation pathway.

    OpenAIRE

    Free, P. F.

    2006-01-01

    Although much has been unravelled with regards to the mechanisms of proteolysis of exogenously derived antigen for presentation via histocompatibility class-II (MHC-II), key questions remain unresolved. The exact role of each proteolytic enzyme in this process is not understood. The aspartic proteinase cathepsin E is hypothesised to play an important role. The aim of this study is to examine this by the use of novel aspartic proteinase inhibitors based upon the aspartic proteinase inhibitor p...

  19. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation.

    Science.gov (United States)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-12-30

    Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Cathepsins: Proteases that are vital for survival but can also be fatal.

    Science.gov (United States)

    Patel, Seema; Homaei, Ahmad; El-Seedi, Hesham R; Akhtar, Nadeem

    2018-06-06

    The state of enzymes in the human body determines the normal physiology or pathology, so all the six classes of enzymes are crucial. Proteases, the hydrolases, can be of several types based on the nucleophilic amino acid or the metal cofactor needed for their activity. Cathepsins are proteases with serine, cysteine, or aspartic acid residues as the nucleophiles, which are vital for digestion, coagulation, immune response, adipogenesis, hormone liberation, peptide synthesis, among a litany of other functions. But inflammatory state radically affects their normal roles. Released from the lysosomes, they degrade extracellular matrix proteins such as collagen and elastin, mediating parasite infection, autoimmune diseases, tumor metastasis, cardiovascular issues, and neural degeneration, among other health hazards. Over the years, the different types and isoforms of cathepsin, their optimal pH and functions have been studied, yet much information is still elusive. By taming and harnessing cathepsins, by inhibitors and judicious lifestyle, a gamut of malignancies can be resolved. This review discusses these aspects, which can be of clinical relevance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  1. Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability.

    Science.gov (United States)

    Zwicker, Jeffery D; Diaz, Nicolas A; Guerra, Alfredo J; Kirchhoff, Paul D; Wen, Bo; Sun, Duxin; Carruthers, Vern B; Larsen, Scott D

    2018-06-01

    The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 μM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. TGF-ß Regulates Cathepsin Activation during Normal and Pathogenic Development.

    Science.gov (United States)

    Flanagan-Steet, Heather; Christian, Courtney; Lu, Po-Nien; Aarnio-Peterson, Megan; Sanman, Laura; Archer-Hartmann, Stephanie; Azadi, Parastoo; Bogyo, Matthew; Steet, Richard A

    2018-03-13

    Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-ß signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-ß-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-ß signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis.

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    Urša Pečar Fonović

    Full Text Available Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1-clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.

  4. Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

    Science.gov (United States)

    Pečar Fonović, Urša; Kos, Janko

    2015-01-01

    Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1—clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1. PMID:26325675

  5. Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhang Dongwei

    2011-11-01

    Full Text Available Abstract Background Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases. Methods We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1 on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-β1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-β1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-β1. Results Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39% and K (41%-76% and a 30% decrease in TGF-β1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-β1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor. Conclusion This study demonstrates that

  6. Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCF10A neoT cells

    International Nuclear Information System (INIS)

    Premzl, J.; Kos, J.

    2003-01-01

    Background. Lysosomal cathepsins B and D have been reported to play a role in various processes leading to progression of malignant disease. In ras-transformed MCF10A neoT cells both enzymes show similar vesicular distribution in perinuclear and peripheral cytoplasmic regions. Results. The co-localization of cathepsins B and D in some vesicles as defined by confocal microscopy supports their co-ordinate activity in the proteolytic cascade. On the other hand, we showed that stefin A, an endogenous intracellular inhibitor of cysteine proteases, did not co-localize with cathepsin B and is presumably not involved in regulation of its enzymatic activity within the vesicles. Intracellular localization of both enzymes was confined to similar vesicles as the fluorescent degradation products of DQ-collagen IV either in individual cells or cell spheroids. The capability of these two enzymes to degrade collagen and other components of extracellular matrix is further supported by the results of Matrigel invasion assay. We showed that specific intracellular (CA-074 Me) and extracellular (CA-074) inhibitors of cathepsin B and pepstatin A, an inhibitor of cathepsin D, significantly reduced invasion of MCF10A neoT cells. Our results also show that in contrast to some other studies the activation peptide of pro-cathepsin D exhibited no mitogenic effect on MCF10A neoT, MCF-7 or HEK-293 cells. Conclusion. We conclude that lysosomal cysteine proteases cathepsins B and D predominantly participate in degradation of extracellular matrix and facilitate invasion of tumour cells. (author)

  7. Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Therese Featherston

    2017-07-01

    Full Text Available AimWe have previously demonstrated the putative presence of two cancer stem cell (CSC subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC, which express components of the renin–angiotensin system (RAS. In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC.Methods3,3-Diaminobenzidine (DAB and immunofluorescent (IF immunohistochemical (IHC staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC.ResultsIHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G.ConclusionThe presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

  8. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. In Vivo Molecular Imaging of Cathepsin and Matrix Metalloproteinase Activity Discriminates between Arthritic and Osteoarthritic Processes in Mice

    Directory of Open Access Journals (Sweden)

    Eline A. Vermeij

    2014-01-01

    Full Text Available Rheumatoid arthritis (RA and osteoarthritis (OA are serologically and clinically distinctive, but at the local level, both diseases have many molecular pathways in common. In vivo molecular imaging can unravel the local pathologic processes involved in both diseases. In this study, we investigated matrix metalloproteinase (MMP and cathepsin activity during cartilage destruction, in an RA and an OA mouse model, using biophotonic imaging of substrate-based probes. Mice with collagen-induced arthritis (CIA or destabilization of the medial meniscus (DMM were imaged using near-infrared fluorescent probes, activated by several cathepsins or MMPs. Fluorescence signal intensity was compared to synovial gene expression, histology, and cartilage staining of a neoepitope of aggrecan cleaved by MMPs with the amino acids DIPEN. Increased cathepsin and MMP activity was seen during CIA, whereas the DMM model only showed increased MMP activity. DIPEN expression was seen only during CIA. A possible explanation can be differences in gene expressions; MMP3 and -13, known to produce DIPEN neoepitopes, were upregulated in the CIA model, whereas MMP12, known to be involved in elastin degradation and chemokine inhibition, was upregulated in the DMM model. Thus, molecular imaging showed no cathepsin activity at the time of cartilage damage in the DMM model, whereas both cathepsins and MMPs are active in the CIA model during disease progression.

  10. Impact of the Enhanced Permeability and Retention (EPR Effect and Cathepsins Levels on the Activity of Polymer-Drug Conjugates

    Directory of Open Access Journals (Sweden)

    Amit K. Rajora

    2014-08-01

    Full Text Available Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR effect and (ii the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B. Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates. An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer, although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.

  11. Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study

    Directory of Open Access Journals (Sweden)

    Carina Mucciolo Melo

    Full Text Available CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group. DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the tumor. There was a significant increase in heparanase-1 and cathepsin B activity in the patients' plasma. CONCLUSION: Overexpression of heparanase-1 and heparanase-2, along with increased heparanase-1 and cathepsin B activity in plasma, is associated with the diagnosis of gastrointestinal carcinoma. These findings provide support for using non-invasive assays (plasma samples as an auxiliary method for diagnosing gastrointestinal tumors.

  12. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: Defective nuclear transport of the virions

    International Nuclear Information System (INIS)

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro

    2006-01-01

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBmΔ64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells

  13. Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

    Science.gov (United States)

    Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

    2016-02-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

  14. Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus.

    Science.gov (United States)

    Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Lv, Peng; Lian, Chaoqun; Chen, Keping

    2013-05-01

    Orf91 (Bm91) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes a predicted 105-amino-acid protein, but its function remains unknown. In the current study, 5'-RACE revealed that the transcription initiation site of Bm91 was - 12 nucleotides upstream of the start codon ATG, transcription of Bm91 was detected from 12 to 96 h postinfection (p.i.) and Bm91 protein was detected from 24 to 96 h p.i. in BmNPV-infected BmN cells. Furthermore, Western blot analysis revealed that Bm91 was in occlusion-derived virus (ODV) but not in budded virus (BV). To investigate the role of Bm91 in baculovirus life cycle, a Bm91-knockout virus was constructed by bacmid recombination in E. coli. Fluorescence and light microscopy showed that the production of BV and occlusion bodies (OBs) in Bm91-deficient-virus-infected BmN cells were similar to those in wild-type-virus-infected ones. Bioassay results showed that genetic deletion of Bm91 did not significantly affect BmNPV infectivity, but extended the median lethal time (LT50). Taken together, these results indicate that Bm91 is not essential for viral propagation in vitro, but absence of the gene may affect the virulence of ODVs in silkworm larvae. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

    Science.gov (United States)

    Xiang, Xingwei; Chen, Lin; Guo, Aiqin; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

    2011-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Characterization of Bombyx mori nucleopolyhedrovirus orf68 gene that encodes a novel structural protein of budded virus.

    Science.gov (United States)

    Iwanaga, Masashi; Kurihara, Masaaki; Kobayashi, Masahiko; Kang, WonKyung

    2002-05-25

    All lepidopteran baculovirus genomes sequenced to date encode a homolog of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf68 gene, suggesting that it performs an important role in the virus life cycle. In this article we describe the characterization of BmNPV orf68 gene. Northern and Western analyses demonstrated that orf68 gene was expressed as a late gene and encoded a structural protein of budded virus (BV). Immunohistochemical analysis by confocal microscopy showed that ORF68 protein was localized mainly in the nucleus of infected cells. To examine the function of orf68 gene, we constructed orf68 deletion mutant (BmD68) and characterized it in BmN cells and larvae of B. mori. BV production was delayed in BmD68-infected cells. The larval bioassays also demonstrated that deletion of orf68 did not reduce the infectivity, but mutant virus took 70 h longer to kill the host than wild-type BmNPV. In addition, dot-blot analysis showed viral DNA accumulated more slowly in mutant infected cells. Further examination suggested that BmD68 was less efficient in entry and budding from cells, although it seemed to possess normal attachment ability. These results suggest that ORF68 is a BV-associated protein involved in secondary infection from cell-to-cell. (c) 2002 Elsevier Science (USA).

  17. Co-chaperone BAG2 Determines the Pro-oncogenic Role of Cathepsin B in Triple-Negative Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kyung-Min Yang

    2017-12-01

    Full Text Available Summary: Triple-negative breast cancer (TNBC is considered incurable with currently available treatments, highlighting the need for therapeutic targets and predictive biomarkers. Here, we report a unique role for Bcl-2-associated athanogene 2 (BAG2, which is significantly overexpressed in TNBC, in regulating the dual functions of cathepsin B as either a pro- or anti-oncogenic enzyme. Silencing BAG2 suppresses tumorigenesis and lung metastasis and induces apoptosis by increasing the intracellular mature form of cathepsin B, whereas BAG2 expression induces metastasis by blocking the auto-cleavage processing of pro-cathepsin B via interaction with the propeptide region. BAG2 regulates pro-cathepsin B/annexin II complex formation and facilitates the trafficking of pro-cathespin-B-containing TGN38-positive vesicles toward the cell periphery, leading to the secretion of pro-cathepsin B, which induces metastasis. Collectively, our results uncover BAG2 as a regulator of the oncogenic function of pro-cathepsin B and a potential diagnostic and therapeutic target that may reduce the burden of metastatic breast cancer. : The mechanisms controlling the pro- and anti-oncogenic roles of cathepsin B are unclear. Yang et al. find that BAG2 is a regulator of the dual functions of its client protein, CTSB, facilitating the progression of TNBC. Keywords: BAG2, cathepsin B, TNBC, tumorigenesis, metastasis, breast cancer, TGN38

  18. Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits

    Directory of Open Access Journals (Sweden)

    Oswald Julia

    2011-07-01

    Full Text Available Abstract Background Cathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS. Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant. Results Cathepsin K messenger RNA was detectable in all brain regions of wild type (WT mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk-/- animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk-/- mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of

  19. Cathepsin B-sensitive polymers for compartment-specific degradation and nucleic acid release.

    Science.gov (United States)

    Chu, David S H; Johnson, Russell N; Pun, Suzie H

    2012-02-10

    Degradable cationic polymers are desirable for in vivo nucleic acid delivery because they offer significantly decreased toxicity over non-degradable counterparts. Peptide linkers provide chemical stability and high specificity for particular endopeptidases but have not been extensively studied for nucleic acid delivery applications. In this work, enzymatically degradable peptide-HPMA copolymers were synthesized by RAFT polymerization of HPMA with methacrylamido-terminated peptide macromonomers, resulting in polymers with low polydispersity and near quantitative incorporation of peptides. Three peptide-HPMA copolymers were evaluated: (i) pHCathK(10), containing peptides composed of the linker phe-lys-phe-leu (FKFL), a substrate of the endosomal/lysosomal endopeptidase cathepsin B, connected to oligo-(L)-lysine for nucleic acid binding, (ii) pHCath(D)K(10), containing the FKFL linker with oligo-(D)-lysine, and (iii) pH(D)Cath(D)K(10), containing all (D) amino acids. Cathepsin B degraded copolymers pHCathK(10) and pHCath(D)K(10) within 1 h while no degradation of pH(D)Cath(D)K(10) was observed. Polyplexes formed with pHCathK(10) copolymers show DNA release by 4 h of treatment with cathepsin B; comparatively, polyplexes formed with pHCath(D)K(10) and pH(D)Cath(D)K(10) show no DNA release within 8 h. Transfection efficiency in HeLa and NIH/3T3 cells were comparable between the copolymers but pHCathK(10) was less toxic. This work demonstrates the successful application of peptide linkers for degradable cationic polymers and DNA release. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Cathepsin activities and thermal properties of Nile tilapia (Oreochromis niloticus meat during ambient storage

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    Tulakhun Nonthaput

    2017-06-01

    Full Text Available Understanding the postmortem changes at ambient aquatic temperature can be useful for estimating the time of death in environmental forensic studies when little information is available. Muscle degradation was investigated in Nile tilapia (Oreochromis niloticus in terms of the specific activities of cathepsins (B, H and L and the scavenging activities and thermal transition properties of myosin and actin, to assess postmortem changes with time (0, 1, 2, 4, 8, 12, 24 and 48 h after death. The study results are relevant to ambient temperatures in Thailand, (about 30 °C. The specific activities of the three cathepsin enzymes increased significantly with postmortem time (p < 0.05 and had a highly significant positive relationship (r = 0.987−0.997, p < 0.01, n = 32. Cathepsin H had the lowest specific activity and exhibited a different type of time profile. Its lowest specific activity was observed at 8 h, which indicated a significant role at this point in time after death. The radical scavenging activities substantially decreased with the time since death, especially within the first 1 h, while no changes occurred from 2 to 8 h, or from 12 to 24 h. The thermal properties of myosin and actin were observed up to a 24 h delay. The degradation of each protein fluctuated with the delay time; actin was more sensitive to postmortem delay than myosin. Overall, the findings from the current study might be used as primary data to estimate the time of death of an aquatic animal. A potential application is for environmental forensics in relation to fish kill events associated with pollution crimes or the mass death of exported fish under transportation insurance, as well as in animal cruelty investigations.

  1. EKSTRAKSI DAN KARAKTERISASI PARSIAL EKSTRAK KASAR ENZIM KATEPSIN DARI IKAN PATIN [Extraction and Partial Characterization of Crude Enzymes Cathepsin from Catfish

    Directory of Open Access Journals (Sweden)

    Muhammad Zakiyul Fikri*

    2014-06-01

    Full Text Available Decomposition of protein by enzymatic process will lead to changes in odor, texture, and appearance of fish. The enzymes that play a role in the enzymatic process is primarily proteolytic enzymes. Cathepsin is one of the proteolytic enzymes found in animal tissue that hydrolyzes peptide bonds of proteins. This study aims to extract the cathepsin, characterize the crude extract derived from catfish. The stages of this research consist of the extraction and characterization of the cathepsin from catfish. Result of the extraction was crude extract of cathepsin with activity of 0.278 U/mL. The enzyme had optimum temperature of 50°C, pH 6 and substrate concentration of 2%. The activity of the cathepsin was inhibited by metal ions of Fe3+, Cu2+, Ca2+, but increased by metal ions of Mg2+.

  2. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

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    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  3. Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L.

    Science.gov (United States)

    Sansri, Veerawat; Meemon, Krai; Changklungmoa, Narin; Kueakhai, Pornanan; Chantree, Pathanin; Chaichanasak, Pannigan; Lorsuwannarat, Natcha; Itagaki, Tadashi; Sobhon, Prasert

    2015-03-24

    Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its

  4. Cathepsin B inhibitory activities of three new phthalate derivatives isolated from seahorse, Hippocampus Kuda Bleeler.

    Science.gov (United States)

    Li, Yong; Qian, Zhong-Ji; Kim, Se-Kwon

    2008-12-01

    Three new phthalate acid derivatives, 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecyl phthalate (1), 2-ethyldecyl 2-ethylundecyl phthalate (2), and bis(2-ethyldodecyl) phthalate (3), were isolated from seahorse, Hippocampus Kuda Bleeler, together with a known natural analog bis(2-ethylheptyl) phthalate (4). The structures of these compounds were elucidated mainly by means of the comprehensive analysis of their NMR spectroscopic data. The four phthalate derivatives showed dose-dependent cathepsin B inhibitions activities with IC(50) values of 0.13 mM (1), 0.21 mM (2), 0.18 mM (3), and 0.29 mM (4), respectively.

  5. IrCL1-The haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus

    Czech Academy of Sciences Publication Activity Database

    Franta, Zdeněk; Sojka, Daniel; Frantová, Helena; Dvořák, Jan; Horn, Martin; Srba, Jindřich; Talacko, Pavel; Mareš, Michael; Schneider, E.; Craik, C. S.; McKerrow, J. H.; Caffrey, C. R.; Kopáček, Petr

    2011-01-01

    Roč. 41, č. 12 (2011), 1253-1262 ISSN 0020-7519 R&D Projects: GA AV ČR IAA600960910; GA ČR(CZ) GAP207/10/2183; GA ČR GPP502/11/P682; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : tick gut * hemoglobin digestion * cathepsin L Subject RIV: EC - Immunology Impact factor: 3.393, year: 2011

  6. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    OpenAIRE

    Breuckmann, Frank; von Kobyletzki, Gregor; Avermaete, Annelies; Kreuter, Alexander; Altmeyer, Peter; Gambichler, Thilo

    2002-01-01

    Abstract Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great value within the treatment of severe atopic dermatitis (AD). The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with...

  7. Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets

    Czech Academy of Sciences Publication Activity Database

    Sojka, Daniel; Hartmann, David; Bartošová-Sojková, Pavla; Dvořák, Jan

    2016-01-01

    Roč. 32, č. 9 (2016), s. 708-723 ISSN 1471-4922 R&D Projects: GA ČR GA14-33693S; GA ČR GA13-11043S; GA ČR(CZ) GAP302/11/1481 EU Projects: European Commission(XE) 248642 - SCHISTOSOMA PROTEASE Institutional support: RVO:60077344 ; RVO:68378050 Keywords : aspartic peptidases * cathepsin D * hemoglobinolysis * parasites * vectors Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 6.333, year: 2016

  8. The synthesis of a tritium, carbon-14, and stable isotope-labeled cathepsin C inhibitors.

    Science.gov (United States)

    Allen, Paul; Bragg, Ryan A; Caffrey, Moya; Ericsson, Cecilia; Hickey, Michael J; Kingston, Lee P; Elmore, Charles S

    2017-02-01

    As part of a medicinal chemistry program aimed at developing a highly potent and selective cathepsin C inhibitor, tritium, carbon-14, and stable isotope-labeled materials were required. The synthesis of tritium-labeled methanesulfonate 5 was achieved via catalytic tritiolysis of a chloro precursor, albeit at a low radiochemical purity of 67%. Tritium-labeled AZD5248 was prepared via a 3-stage synthesis, utilizing amide-directed hydrogen isotope exchange. Carbon-14 and stable isotope-labeled AZD5248 were successfully prepared through modifications of the medicinal chemistry synthetic route, enabling the use of available labeled intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Dose responses of in vivo- and in vitro-produced strains of gypsy moth (Lepidoptera: Lymantriidae) nucleopolyhedrovirus (LdMNPV) applied with and without the virus enhancer Blankophor BBH

    Science.gov (United States)

    John D. Podgwaite; James M. Slavicek; Kevin W. Thorpe; Ralph E. Webb; Roger W. Fuester; Vincent D' Amico; Randel A. Peiffer; Michael A. Valenti

    2013-01-01

    The gypsy moth, Lymantria dispar L., nucleopolyhedrovirus (LdMNPV) product Gypchek is a microbial pesticide produced by the USDA Forest Service. Gypchek is a mixture of LdMNPV genotypes produced in vivo. Commercial interests prefer to develop a stable, high-potency genotype that can be produced at low cost, preferably in vitro. We sprayed 2 LdMNPV...

  10. Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination1[W

    Science.gov (United States)

    Martinez, Manuel; Cambra, Ines; Carrillo, Laura; Diaz-Mendoza, Mercedes; Diaz, Isabel

    2009-01-01

    Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds. PMID:19759340

  11. A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

    Science.gov (United States)

    Godahewa, G I; Perera, N C N; Lee, Sukkyoung; Kim, Myoung-Jin; Lee, Jehee

    2017-09-05

    Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone. Copyright © 2017. Published by Elsevier

  12. Effect of Individual and Combined Treatment with Azadirachtin and Spodoptera littoralis Multicapsid Nucleopolyhedrovirus (SpliMNPV, Baculoviridae on the Egyptian Cotton Leafworm Spodoptera littoralis (Boisduval (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    El-Sayed H. Shaurub

    2014-12-01

    Full Text Available The tetranortriterpenoid, azadirachtin, and the entomopathogenic virus, nucleopolyhedrovirus, are used as safe and new control measures for combating agricultural insect pests instead of the use of synthetic insecticides. They can be mixed together as an integrated pest management strategy. Thus, the current investigation was designed to determine the mortality, duration and weight gain of Spodoptera littoralis (Boisduval (Lepidoptera: Noctuidae larvae, and the yield of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV (Baculoviridae when the fourth larval instars were treated individually with the LC50 of azadirachtin and of SpliMNPV, and in combination with each other using the LC25, compared to non-treated larvae (control. The results obtained showed that combined treatment significantly enhanced the larval mortality by about 58.40 %, i.e. potentiation. Both individual and combined treatment significantly decreased the larval weight gain, whereas the larval duration was significantly increased, with the highest change in case of combined treatment. Azadirachtin–NPV mixture significantly decreased the viral yield (number of polyhedral inclusion bodies/g fresh larval body weight by about 36.05 % compared to the individual treatment with the NPV. It can be concluded that although azadirachtin enhanced the pathogenicity (% larval kill of SpliMNPV to S. littoralis, azadirachtin–SpliMNPV mixture is unlikely to be useful for the mass production of this viral isolate. Thus, these laboratory observations require validation in field studies under commercial growing conditions.

  13. Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid.

    Science.gov (United States)

    Sun, Jingchen; Yao, Lunguang; Yao, Ning; Xu, Hua; Jin, Pengfei; Kan, Yunchao

    2010-12-01

    The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1 ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.

  14. The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

    Science.gov (United States)

    Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

    1998-03-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

  15. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  16. Molecular cloning and functional characterization of cathepsin D from sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Qin, Lei; Du, Ming

    2017-11-01

    Cathepsin D (CTSD, EC 3.4.23.5) belongs to aspartic protease family, which is located in lysosomes and is distributed in diverse tissues and cells. CTSD has a wide variety of physiological functions, owing to its proteolytic activity in degradating proteins and peptides. In the current study, the full length cDNA of sea cucumber (Apostichopus japonicus) cathepsin D (AjCTSD) was firstly cloned, then the association between AjCTSD and sea cucumber autolysis was investigated. The full length cDNA of AjCTSD was 2896 bp, with an open reading frame (ORF) for 391 amino acids. AjCTSD was widely expressed in body wall, muscle and intestine; the expression level was the highest in intestine, followed by muscle and body wall. Compared to fresh tissues, AjCTSD expression levels were significantly increased in all examined autolytic tissues. The purified recombinant AjCTSD promoted the degradation of sea cucumber muscle. In conclusion, AjCTSD contributed to sea cucumber muscle autolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Crystallographic, DFT and docking (cathepsin B) studies on an organotellurium(IV) compound

    International Nuclear Information System (INIS)

    Caracelli, Ignez; Maganhi, Stella H.; Zukerman-Schpector, Julio; Sousa Madureira, Lucas; Stefani, Helio A.; Guadagnin, Rafael C.; Tiekink, Edward R.T.

    2016-01-01

    Some biologically active organotellurium compounds exhibit inhibitory potency against cathepsin B. In this study, an alkyl derivative, viz. [CH 3 (CH 2 ) 2 C(I)=C(H)](nBu)TeI 2 , 1, has been structurally characterised by X-ray crystallography and shown to be coordinated within a C 2 I 2 donor set. When the stereochemically active lone pair of electrons is taken into account, a distorted trigonal bipyramidal geometry results with the iodide atoms in axial positions. Both intra- and inter-molecular Te..I interactions are also noted. If all interactions are considered, the coordination geometry is based on a Ψ-pentagonal bipyramidal geometry. An unusual feature of the structure is the curving of the functionalised C 5 chain. This feature has been explored by DFT methods and shown to arise as a result of close C-H..I interactions. A docking study (cathepsin B) was performed to understand the inhibition mechanism and to compare the new results with previous observations. Notably, 1 has the same pose exhibited by analogous biologically active compounds with aryl groups. Thus, the present study suggests that (alkyl) 2 TeX 2 compounds should also be evaluated for biological activity.

  18. Crystallographic, DFT and docking (cathepsin B) studies on an organotellurium(IV) compound

    Energy Technology Data Exchange (ETDEWEB)

    Caracelli, Ignez; Maganhi, Stella H. [Univ. Federal de Sao Carlos (Brazil). BioMat; Zukerman-Schpector, Julio; Sousa Madureira, Lucas [Univ. Federal de Sao Carlos (Brazil). Lab. de Cristalografia, Estereodinamica e Modelagem Molecular; Stefani, Helio A. [Sao Paulo Univ. (Brazil). Dept. de Farmacia; Guadagnin, Rafael C. [Univ. Federal de Sao Paulo, Diadema (Brazil). Inst. e Ciencias Mabientais, Quimicas e Farmaceuticas; Tiekink, Edward R.T. [Sunway Univ., Selangor Darul Ehsan (Malaysia). Centre for Crystalline Materials

    2016-08-01

    Some biologically active organotellurium compounds exhibit inhibitory potency against cathepsin B. In this study, an alkyl derivative, viz. [CH{sub 3}(CH{sub 2}){sub 2}C(I)=C(H)](nBu)TeI{sub 2}, 1, has been structurally characterised by X-ray crystallography and shown to be coordinated within a C{sub 2}I{sub 2} donor set. When the stereochemically active lone pair of electrons is taken into account, a distorted trigonal bipyramidal geometry results with the iodide atoms in axial positions. Both intra- and inter-molecular Te..I interactions are also noted. If all interactions are considered, the coordination geometry is based on a Ψ-pentagonal bipyramidal geometry. An unusual feature of the structure is the curving of the functionalised C{sub 5} chain. This feature has been explored by DFT methods and shown to arise as a result of close C-H..I interactions. A docking study (cathepsin B) was performed to understand the inhibition mechanism and to compare the new results with previous observations. Notably, 1 has the same pose exhibited by analogous biologically active compounds with aryl groups. Thus, the present study suggests that (alkyl){sub 2}TeX{sub 2} compounds should also be evaluated for biological activity.

  19. Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica.

    Science.gov (United States)

    Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

    2012-12-01

    Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

    Science.gov (United States)

    Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

    2010-01-01

    Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

  1. Cathepsin B Expression and the Correlation with Clinical Aspects of Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Yang, Wei-En; Ho, Chuan-Chen; Yang, Shun-Fa; Lin, Shu-Hui; Yeh, Kun-Tu; Lin, Chiao-Wen; Chen, Mu-Kuan

    2016-01-01

    Cathepsin B (CTSB), a member of the cathepsin family, is a cysteine protease that is widely distributed in the lysosomes of cells in various tissues. It is overexpressed in several human cancers and may be related to tumorigenesis. The main purpose of this study was to analyze CTSB expression in oral squamous cell carcinoma (OSCC) and its correlation with patient prognosis. Tissue microarrays were used to detect CTSB expression in 280 patients and to examine the association between CTSB expression and clinicopathological parameters. In addition, the metastatic effects of the CTSB knockdown on two oral cancer cell lines were investigated by transwell migration assay. Cytoplasmic CTSB expression was detected in 34.6% (97/280) of patients. CTSB expression was correlated with positive lymph node metastasis (p = 0.007) and higher tumor grade (p = 0.008) but not with tumor size and distant metastasis. In addition, multivariate analysis using a Cox proportional hazards model revealed a higher hazard ratio, demonstrating that CTSB expression was an independent unfavorable prognostic factor in buccal mucosa carcinoma patients. Furthermore, the Kaplan-Meier curve revealed that buccal mucosa OSCC patients with positive CTSB expression had significantly shorter overall survival. Moreover, treatment with the CTSB siRNA exerted an inhibitory effect on migration in OC2 and CAL27 oral cancer cells. We conclude that CTSB expression may be useful for determining OSCC prognosis, particularly for patients with lymph node metastasis, and may function as a biomarker of the survival of OSCC patients in Taiwan.

  2. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

    2006-01-09

    Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

  3. Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation.

    Science.gov (United States)

    Oh, Sang-Seok; Park, Soojong; Lee, Ki-Won; Madhi, Hamadi; Park, Sae Gwang; Lee, Hee Gu; Cho, Yong-Yeon; Yoo, Jiyun; Dong Kim, Kwang

    2017-04-06

    Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

  4. Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization.

    Science.gov (United States)

    Oresic Bender, Kristina; Ofori, Leslie; van der Linden, Wouter A; Mock, Elliot D; Datta, Gopal K; Chowdhury, Somenath; Li, Hao; Segal, Ehud; Sanchez Lopez, Mateo; Ellman, Jonathan A; Figdor, Carl G; Bogyo, Matthew; Verdoes, Martijn

    2015-04-15

    The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

  5. Tumor Necrosis Factor-α Induced Apoptosis in U937 Cells Promotes Cathepsin D-Independent Stefin B Degradation.

    Science.gov (United States)

    Bidovec, Katja; Božič, Janja; Dolenc, Iztok; Turk, Boris; Turk, Vito; Stoka, Veronika

    2017-12-01

    Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

    International Nuclear Information System (INIS)

    Yoon, J-Y; Szwajcer, D; Ishdorj, G; Benjaminson, P; Xiao, W; Kumar, R; Johnston, J B; Gibson, S B

    2013-01-01

    Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins

  7. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  8. Single- and Double-Headed Chemical Probes for Detection of Active Cathepsin D in a Cancer Cell Proteome

    Czech Academy of Sciences Publication Activity Database

    Nussbaumerová, Martina; Srp, Jaroslav; Máša, Martin; Hradilek, Martin; Šanda, Miloslav; Reiniš, Milan; Horn, Martin; Mareš, Michael

    2010-01-01

    Roč. 11, č. 11 (2010), s. 1538-1541 ISSN 1439-4227 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : cathepsin D * cancer * activity-based probes Subject RIV: CE - Biochemistry Impact factor: 3.945, year: 2010

  9. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  10. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.

    Science.gov (United States)

    Zhang, T; Rawson, D M; Tosti, L; Carnevali, O

    2008-04-01

    This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk

  11. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  12. Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

    Directory of Open Access Journals (Sweden)

    Florencia Ferraro

    2016-07-01

    Full Text Available Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections.We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1. Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 μM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells.Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34 is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control

  13. A cathepsin L-like protease from Strongylus vulgaris: an orthologue of Caenorhabditis elegans CPL-1.

    Science.gov (United States)

    Ultaigh, Sinéad Nic An; Carolan, James C; Britton, Collette; Murray, Linda; Ryan, Michael F

    2009-04-01

    Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditis elegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylus vulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgaris cpl-1 rescued the embryonic lethal phenotype of the C.elegans cpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.

  14. Purification and characterization of cathepsin D from herring muscle ( Clupea harengus )

    DEFF Research Database (Denmark)

    Nielsen, L.B.; Nielsen, Henrik Hauch

    2001-01-01

    hamatus) and trout ovary (Oncorhynchus mykiss). Digestion of the P-chain of oxidized insulin resulted in preferential cleavage at Leu(15)-Tyr(16), (47%), Tyr(16)-Leu(17) (34%) and Ala(14)- Leu(15) (18%). Incubation with myofibrils from herring muscle at pH 4.23 showed that the enzyme mainly degraded......Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38 000-39 000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at p...

  15. Targeting Cathepsin E in Pancreatic Cancer by a Small Molecule Allows In Vivo Detection

    Directory of Open Access Journals (Sweden)

    Edmund J. Keliher

    2013-07-01

    Full Text Available When resectable, invasive pancreatic ductal adenocarcinoma (PDAC is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE is overexpressed in PDAC and that an United States Food and Drug Administration (FDA-approved protease inhibitor has high affinity for CTSE, we have developed a CTSE optical imaging agent [ritonavir tetramethyl-BODIPY (RIT-TMB] for potential intraoperative use.We show nanomolar affinity [half maximal inhibitory concentration (IC50 of 39.9 ± 1.2 nM] against CTSE of the RIT-TMB in biochemical assays and intracellular accumulation and target-to-background ratios that allow specific delineation of individual cancer cells. This approach should be useful for more refined surgical staging, planning, and resection with curative intent.

  16. E2-mediated cathepsin D (CTSD) activation involves looping of distal enhancer elements.

    Science.gov (United States)

    Bretschneider, Nancy; Kangaspeska, Sara; Seifert, Martin; Reid, George; Gannon, Frank; Denger, Stefanie

    2008-08-01

    Estrogen receptor alpha (ERalpha) is a ligand dependent transcription factor that regulates the expression of target genes through interacting with cis-acting estrogen response elements (EREs). However, only a minority of ERalpha binding sites are located within the proximal promoter regions of responsive genes. Here we report the characterization of an ERE located 9kbp upstream of the TSS of the cathepsin D gene (CTSD) that up-regulates CTSD expression upon estrogen stimulation in MCF-7 cells. Using ChIP, we show recruitment of ERalpha and phosphorylated PolII at the CTSD distal enhancer region. Moreover, we determine the kinetics of transient CpG methylation on the promoter region of CTSD and for the first time, at a distal enhancer element. We show that ERalpha is crucial for long-distance regulation of CTSD expression involving a looping mechanism.

  17. Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.

    Science.gov (United States)

    Visessanguan, Wonnop; Benjakul, Soottawat; An, Haejung

    2003-03-01

    A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.

  18. Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection.

    Science.gov (United States)

    Chen, Lin; Shen, Yunwang; Yang, Rui; Wu, Xiaofeng; Hu, Wenjun; Shen, Guoxin

    2015-10-24

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf64 (Bm64, a homologue of ac78) is a core baculovirus gene. Recently, Li et al. reported that Ac78 was not essential for budded viruses (BVs) production and occlusion-derived viruses (ODVs) formation (Virus Res 191:70-82, 2014). Conversely, Tao et al. demonstrated that Ac78 was localized to the BV and ODV envelopes and was required for BV production and ODV formation (J Virol 87:8441-50, 2013). In this study, the function of Bm64 was characterized to determine the role of Bm64 in the BmNPV infection cycle. The temporal expression of Bm64 was examined using total RNA extracted from BmNPV-infected BmN cells at different time points by reverse-transcription PCR (RT-PCR) and 5' RACE analysis. To determine the functions of Bm64 in viral replication and the viral phenotype throughout the viral life cycle, a deletion virus (vBm(64KO)) was generated via homologous recombination in Escherichia coli. Viral replication and BV production were determined by real-time PCR. Electron microscopy was used to detect virion morphogenesis. The subcellular localization of Bm64 was determined by microscopy, and per os infectivity was used to determine its role in the baculovirus oral infection cycle. Viral plaque and titer assay results showed that a few infectious BVs were produced by vBm(64KO), suggesting that deletion of Bm64 affected BV production. Viral DNA replication was detected and polyhedra were observed in vBm(64KO)-transfected cells. Microscopy analysis revealed that Bm64 was predominantly localized to the ring zone of the nuclei during the infection cycle. Electron microscopy showed that Bm64 was not essential for the formation of ODVs or the subsequent occlusion of ODV into polyhedra. The per os infectivity results showed that the polyhedra of vBm(64KO) were unable to infect silkworm larvae. In conclusion, our results suggest that Bm64 plays an important role in BV production and per os infection, but is not required for viral DNA

  19. Cathepsin D immobilized capillary reactors for on-flow screening assays.

    Science.gov (United States)

    Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

    2018-03-20

    The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica.

    Science.gov (United States)

    Sansri, Veerawat; Changklungmoa, Narin; Chaichanasak, Pannigan; Sobhon, Prasert; Meemon, Krai

    2013-10-01

    Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection. Copyright

  1. The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

    Science.gov (United States)

    Chen, W; Gao, B; Hao, L; Zhu, G; Jules, J; MacDougall, M J; Wang, J; Han, X; Zhou, X; Li, Y-P

    2016-10-01

    Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci.

    Directory of Open Access Journals (Sweden)

    Martin A Bewley

    2011-01-01

    Full Text Available The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

  3. Cathepsin C and plasma glutamate carboxypeptidase secreted from Fischer rat thyroid cells liberate thyroxin from the N-terminus of thyroglobulin.

    Science.gov (United States)

    Suban, Dejan; Zajc, Tajana; Renko, Miha; Turk, Boris; Turk, Vito; Dolenc, Iztok

    2012-03-01

    The release of a thyroid hormone from thyroglobulin is controlled by a complex regulatory system. We focused on the extracellular action of two lysosomal enzymes, cathepsin C (catC, dipeptidyl peptidase I) and PGCP (lysosomal dipeptidase), on thyroglobulin, and their ability to liberate the hormone thyroxin. Cathepsin C, an exopeptidase, removes dipeptides from the N-terminus of substrates, and PGCP hydrolyses dipeptides to amino acids. In vitro experiments proved that cathepsin C removes up to 12 amino acids from the N-terminus of porcine thyroglobulin, including a dipeptide with thyroxin on position 5. The newly formed N-terminus, Arg-Pro-, was not hydrolysed further by cathepsin C. Cell culture experiments with FRTL-5 cell line showed localization of cathepsin C and PGCP and their secretion into the medium. Secretion of the active cathepsin C from FRTL-5 cells is stimulated by TSH, insulin, and/or somatostatin. The released enzymes liberate thyroxin from porcine thyroglobulin added to media. The hormone liberation can be reduced by synthetic inhibitors of cysteine proteinases and metalloproteinases. Additionally, we show that TSH, insulin, and/or somatostatin induce up-regulation of N-acetylglucosaminyltransferase 1, the enzyme responsible for the initiation of biosynthesis of hybrid and complex N-glycosylation of proteins. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  4. Cysteine peptidases of Eudiplozoon nipponicum: a broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp

    Czech Academy of Sciences Publication Activity Database

    Jedličková, L.; Dvořáková, H.; Dvořák, J.; Kašný, M.; Ulrychová, Lenka; Vorel, J.; Žárský, V.; Mikeš, L.

    2018-01-01

    Roč. 11, Mar 6 (2018), č. článku 142. ISSN 1756-3305 R&D Projects: GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : cysteine peptidase * protease * cathepsin * S2 subsite * haematophagy * blood digestion Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.080, year: 2016 https:// parasites andvectors.biomedcentral.com/ articles /10.1186/s13071-018-2666-2

  5. Development of an SPR imaging biosensor for determination of cathepsin G in saliva and white blood cells

    International Nuclear Information System (INIS)

    Gorodkiewicz, E.; Wojtulewski, K.; Regulska, E.

    2011-01-01

    Cathepsin G (CatG) is an endopeptidase that is associated with the early immune response. The synthetic compound cathepsin G inhibitor I (CGI-I) was tested for its ability to inhibit the activity of CatG via a new surface plasmon resonance imaging assay. CGI-I was immobilized on the gold surface of an SPR sensor that was first modified with 1-octadecanethiol. A concentration of CGI-I equal to 4.0 μg.mL -1 and a pH of 8.0 were found to give the best results. The dynamic response of the sensor ranges from 0. 25 to 1. 5 ng.mL -1 , and the detection limit is 0. 12 ng.mL -1 . The sensor was applied to detect CatG in human saliva and white blood cells. (author)

  6. Distribution of Cathepsin K in Late Stage of Tooth Germ Development and Its Function in Degrading Enamel Matrix Proteins in Mouse.

    Directory of Open Access Journals (Sweden)

    Tao Jiang

    Full Text Available Cathepsin K (CTSK is a member of cysteine proteinase family, and is predominantly expressed in osteoclastsfor degradationof bone matrix proteins. Given the similarity in physical properties of bone and dental mineralized tissues, including enamel, dentin and cementum, CTSK is likely to take part in mineralization process during odontogenesis. On the other hand, patients with pycnodysostosis caused by mutations of the CTSK gene displayedmultipledental abnormalities, such as hypoplasia of the enamel, obliterated pulp chambers, hypercementosis and periodontal disease. Thereforeitis necessary to study the metabolic role of CTSK in tooth matrix proteins. In this study, BALB/c mice at embryonic day 18 (E18, post-natal day 1 (P1, P5, P10 and P20 were used (5 mice at each time pointfor systematic analyses of CTSK expression in the late stage of tooth germ development. We found that CTSK was abundantly expressed in the ameloblasts during secretory and maturation stages (P5 and P10 by immunohistochemistry stainings.During dentinogenesis, the staining was also intense in the mineralization stage (P5 and P10,but not detectable in the early stage of dentin formation (P1 and after tooth eruption (P20.Furthermore, through zymography and digestion test in vitro, CTSK was proved to be capable of hydrolyzing Emdogain and also cleaving Amelogenininto multiple products. Our resultsshed lights on revealing new functions of CTSK and pathogenesis of pycnodysostosis in oral tissues.

  7. Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

    Science.gov (United States)

    Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

    2001-10-01

    Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

  8. Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

    Science.gov (United States)

    Damasceno, Ticiane F; Dias, Renata O; de Oliveira, Juliana R; Salinas, Roberto K; Juliano, Maria A; Ferreira, Clelia; Terra, Walter R

    2017-10-01

    Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

    Science.gov (United States)

    Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

    2012-09-01

    Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

    Directory of Open Access Journals (Sweden)

    Louise Ford

    Full Text Available Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1 and cathepsin Z (Ce-cpz-1 has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

  11. Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

    OpenAIRE

    Gibbons, Aileen M.; McElvaney, Noel; Taggart, Clifford; Cryan, Sally-Ann

    2009-01-01

    Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-s...

  12. Functional expression and characterization of cathepsin B and L from the gut of the tick Ixodes ricinus

    Czech Academy of Sciences Publication Activity Database

    Franta, Zdeněk; Pěničková, Helena; Dvorak, J.; Schneider, E. I.; Horn, Martin; Mareš, Michael; Sojka, Daniel; McKerrow, J. H.; Caffrey, C. R.; Kopáček, Petr

    2009-01-01

    Roč. 276, S1 (2009), s. 309-309 ISSN 1742-464X. [34th FEBS Congress: Life's Molecular Interactions. 04.07.2009-09.07.2009, Prague] R&D Projects: GA AV ČR IAA600960910; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : cathepsin B and L * Ixodes ricinus * functionl expresssion Subject RIV: EB - Genetics ; Molecular Biology

  13. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Li; Wu, Zhou; Baba, Masashi [Department of Aging Science and Pharmacology, Faculty of Dental Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582 (Japan); Peters, Christoph [Institute fuer Molekulare Medizin und Zellforshung, Albert-Ludwings-Universitaet Freiburg, D-79104 Freiburg (Germany); Uchiyama, Yasuo [Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo (Japan); Nakanishi, Hiroshi, E-mail: nakan@dent.kyushu-u.ac.jp [Department of Aging Science and Pharmacology, Faculty of Dental Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582 (Japan)

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  14. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    International Nuclear Information System (INIS)

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-01-01

    Research highlights: → Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. → CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. → CB-deficiency significantly increased the mean survival ratio of injured neurons. → Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy-induced mortor neuron

  15. Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.

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    Tien-Sheng Tseng

    Full Text Available Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB. Far-UV circular dichroism (CD, intrinsic fluorescence, and nuclear magnetic resonance (NMR spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.

  16. Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

    Science.gov (United States)

    Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

    2012-03-01

    The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. [Cathepsin K as a biomarker of bone involvement in type 1 Gaucher disease].

    Science.gov (United States)

    Bobillo Lobato, Joaquín; Durán Parejo, Pilar; Núñez Vázquez, Ramiro J; Jiménez Jiménez, Luis M

    2015-10-05

    Gaucher disease is an inherited disorder caused by deficit of acid β-glucocerebrosidase, responsible for the degradation of glucosylceramide to ceramide and glucose. Although the disorder is primarily hematologic, bone is the second most commonly affected structure. Cathepsin K (CATK) is an enzyme involved in bone remodelling process. It has been proposed that determination of its serum concentrations may provide additional information to other biomarkers. The study included 20 control subjects and 20 Gaucher type 1 patients from Andalusia and Extremadura regions. We analyzed the biomarkers of bone remodelling: the bone alkaline phosphatase (B-ALP), the N-terminal propeptide of type 1 procollagen (P1NP), the β carboxyterminal telopeptide of type 1 collagen (CTx) and the CATK through electrochemiluminescence and immunoassay techniques. There is an increase in levels of CATK, CATK/P1NP and CATK/B-ALP ratios in type 1 Gaucher patients compared to the control group. Considering the existence of skeletal manifestations in the patient group, the CATK and CATK/P1NP ratio showed higher levels in patients with bone damage compared to those without it. Although imaging studies are the gold standard for monitoring bone disease in type 1 Gaucher patients, the utility of CATK should be considered as a possible indicator of bone damage in these patients. Furthermore, this parameter can be used in the monitoring of the treatment of bone pathology. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  18. Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy.

    Science.gov (United States)

    Huang, Kun; Gao, Lu; Yang, Ming; Wang, Jiliang; Wang, Zheng; Wang, Lin; Wang, Guobin; Li, Huili

    2017-08-01

    Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Detection of oral squamous cell carcinoma metastasis with cathepsin D: An immunohistochemical approach

    Directory of Open Access Journals (Sweden)

    Seema Kapoor

    2014-01-01

    Full Text Available Background: The lysosomal protease cathepsin D (CD has been associated with tumor progression in malignant tumors including oral squamous cell carcinoma (OSCC. The purpose of this study was to find out any association between the CD and lymph node metastasis and to study the correlation of CD with various clinicopathological parameters to aid in assessment of its role as a prognostic indicator. Materials and Methods: Immunohistochemical staining was performed on 20 OSCC samples with polyclonal antibody against CD. Positive results indicative of the presence of CD were further analyzed to determine any correlation between the CD and other clinicopathological parameters. Pearson Chi-square analyses, Spearsman correlation coefficient, Mann-Whitney test, Kruskal Wallis test and student t test were used for statistical analysis (P < 0.05. Results: Patients with lymph node metastasis showed statistically significant increase in CD expression (P < 0.01. Increasing tumor size seemed to correlate with increased CD expression (P < 0.05. Conclusion: Based on its association with other clinicopathological variables, CD expression can be used for the assessment of patient survival in cases of OSCC.

  20. Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis.

    Science.gov (United States)

    Chen, Wenjun; Ning, Dan; Wang, Xiaoyun; Chen, Tingjin; Lv, Xiaoli; Sun, Jiufeng; Wu, De; Huang, Yan; Xu, Jin; Yu, Xinbing

    2015-12-21

    Human clonorchiasis is a prevailing food-borne disease caused by Clonorchis sinensis infection. Functional characterizations of key molecules from C. sinensis could facilitate the intervention of C. sinensis associated diseases. In this study, immunolocalization of C. sinensis cathepsin B proteases (CsCBs) in C. sinensis worms was investigated. Four CsCBs were expressed in Pichia pastoris yeast cells. Purified yCsCBs were measured for enzymatic and hydrolase activities in the presence of various host proteins. Cell proliferation, wound-healing and transwell assays were performed to show the effect of CsCBs on human cells. CsCBs were localized in the excretory vesicle, oral sucker and intestinal tract of C. sinensis. Recombinant yCsCBs from yeast showed active enzymatic activity at pH 5.0-5.5 and at 37-42 °C. yCsCBs can degrade various host proteins including human serum albumin, human fibronectin, human hemoglobin and human IgG. CsCBs were detected in liver tissues of mice and cancer patients afflicted with clonorchiasis. Various bioassays collectively demonstrated that CsCBs could promote cell proliferation, migration and invasion of human cancer cells. Our results demonstrated that CsCBs can degrade various human proteins and we proved that the secreted CsCBs are involved in the pathogenesis of clonorchiasis.

  1. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  2. Excision of foreign gene product with cathepsin D in chicken hepatoma cell line

    International Nuclear Information System (INIS)

    Sato, Masaharu; Kawashima, Tsuyoshi; Aosasa, Masayoshi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

    2005-01-01

    To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24 h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens

  3. Cathepsin B Improves ß-Amyloidosis and Learning and Memory in Models of Alzheimer's Disease.

    Science.gov (United States)

    Embury, Christine M; Dyavarshetty, Bhagyalaxmi; Lu, Yaman; Wiederin, Jayme L; Ciborowski, Pawel; Gendelman, Howard E; Kiyota, Tomomi

    2017-06-01

    Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD), dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However, whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end, progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels, increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits.

  4. Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

    International Nuclear Information System (INIS)

    Fan, Qi; Wang, Xuedi; Zhang, Hanguang; Li, Chuanwei; Fan, Junhua; Xu, Jing

    2012-01-01

    Highlights: ► Cat S is highly expressed in HCC cells with high metastatic potential. ► Knockdown of Cat S inhibits growth and invasion of HCC cells. ► Knockdown of Cat S inhibits HCC-associated angiogenesis. ► Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

  5. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  6. Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis.

    Directory of Open Access Journals (Sweden)

    David Freeman

    Full Text Available α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

  7. Cathepsin X in serum from patients with colorectal cancer: relation to prognosis

    Science.gov (United States)

    Vizin, Tjasa; Christensen, Ib Jarle; Nielsen, Hans Jørgen; Kos, Janko

    2012-01-01

    Background Up-regulation of lysosomal cysteine protease cathepsin X (Cat X) is associated with disorders of the immune system and neurodegenerative diseases, while its role in the development and progression of cancer is less understood. Enhanced secretion of pro-Cat X was observed in malignant processes, and therefore, the level of total serum Cat X rather than the active enzyme may better reflect the tumour status. Patients and methods Seventy-seven patients with colorectal cancer (CRC) were included in a retrospective study. Blood samples were collected prior to therapy. Using ELISA, the values of total Cat X were measured in serum. Groups of healthy persons (n=77), patients with adenomas (n=77) and patients with non-neoplastic findings (n=77) were included. Results Significant differences between the group of colorectal patients and the groups of healthy persons, adenoma patients and patients with non-malignant findings could not be shown (p=0.89). Within the group of CRC, higher levels of total Cat X significantly correlated to shorter overall survival (HR=2.08, 95% CI:1.07–4.05, p=0.028). Conclusions Total serum Cat X could be a useful prognostic indicator for determining survival of patients with CRC. Increased serum levels of total Cat X may reflect more aggressive tumour cell phenotypes and suggest the involvement of Cat X in processes involved in later stages of tumour progression. PMID:23077459

  8. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

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    Iris J Gonzalez-Leal

    2014-09-01

    Full Text Available Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb and L (Ctsl play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC and macrophages (BMM from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12 expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  9. Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

    Science.gov (United States)

    Siricoon, Sinee; Vichasri Grams, Suksiri; Lertwongvisarn, Kittisak; Abdullohfakeeyah, Muntana; Smooker, Peter M; Grams, Rudi

    2015-12-01

    Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Evaluation of matrix metalloproteinase and cysteine cathepsin activity in dentin hybrid layer by gelatin zymography.

    Science.gov (United States)

    Mahalaxmi, Sekar; Madhubala, Manavalan Madhana; Jayaraman, Mahendran; Sathyakumar, Shanmugasundaram

    2016-01-01

    The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique. Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer's instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/dentin interfaces. These were then ground down to 500 µm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days. Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2. Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2.

  11. Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis.

    Science.gov (United States)

    Chen, Wenjun; Wang, Xiaoyun; Lv, Xiaoli; Tian, Yanli; Xu, Yanquan; Mao, Qiang; Shang, Mei; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2014-09-01

    Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P sinensis excretory/secretory products that may regulate host immune responses.

  12. Pharmacokinetics and pharmacodynamics of the cathepsin S inhibitor, LY3000328, in healthy subjects.

    Science.gov (United States)

    Payne, Christopher D; Deeg, Mark A; Chan, Melanie; Tan, Lai Hock; LaBell, Elizabeth Smith; Shen, Tong; DeBrota, David J

    2014-12-01

    The aim of this study was to assess the safety and tolerability, pharmacokinetics and pharmacodynamics of LY3000328 when administered as single escalating doses to healthy volunteers. This was a phase 1, placebo-controlled, dose escalation study with LY3000328 in 21 healthy male volunteers. Subjects were administered escalating LY3000328 doses up to 300 mg with food in this single dose study. Blood samples were collected at set times post-dose for the assessment of LY3000328 pharmacokinetics and the measurement of cathepsin S (CatS) activity, CatS mass and calculated CatS specific activity. All doses of LY3000328 were well tolerated, with linear pharmacokinetics up to the 300 mg dose. The pharmacodynamic activity of LY3000328 was measured ex vivo showing a biphasic response to LY3000328, where CatS activity declines, then returns to baseline, and then increases to a level above baseline. CatS mass was also assessed post-dose which increased in a dose-dependent manner, and continued to increase after LY3000328 had been cleared from the body. CatS specific activity was additionally calculated to normalize CatS activity for changes in CatS mass. This demonstrated the increase in CatS activity was attributable to the increase in CatS mass detected in plasma. A specific inhibitor of CatS which is cleared quickly from plasma may produce a transient decrease in plasma CatS activity which is followed by a more prolonged increase in plasma CatS mass which may have implications for the future clinical development of inhibitors of CatS. © 2014 The British Pharmacological Society.

  13. Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S.

    Science.gov (United States)

    Galibert, Matthieu; Wartenberg, Mylène; Lecaille, Fabien; Saidi, Ahlame; Mavel, Sylvie; Joulin-Giet, Alix; Korkmaz, Brice; Brömme, Dieter; Aucagne, Vincent; Delmas, Agnès F; Lalmanach, Gilles

    2018-01-20

    Cathepsin (Cat) K is a critical bone-resorbing protease and is a relevant target for the treatment of osteoporosis and bone metastasis, while CatS is an attractive target for drugs in autoimmune diseases (e.g. rheumatoid arthritis), emphysema or neuropathic pain. Despite major achievements, current pharmacological inhibitors are still lacking in safety and may have damaging side effects. A promising strategy for developing safer reversible and competitive inhibitors as new lead compounds could be to insert non-cleavable bonds at the scissile P1-P1' position of selective substrates of CatS and CatK. Accordingly, we introduced a 1,4-disubstituted 1,2,3-triazole heterocycle that mimics most of the features of a trans-amide bond, or we incorporated a semicarbazide bond (azaGly residue) by replacing the α-carbon of the glycyl residue at P1 by a nitrogen atom. AzaGly-containing peptidomimetics inhibited powerfully their respective target proteases in the nM range, while triazolopeptides were weaker inhibitors (Ki in the μM range). The selectivity of the azaGly CatS inhibitor (1b) was confirmed by using spleen lysates from wild-type vs CatS-deficient mice. Alternatively, the azaGly bradykinin-derived CatK inhibitor (2b) potently inhibited CatK (Ki = 9 nM) and impaired its kininase activity in vitro. Molecular modeling studies support that the semicarbazide bond of 2b is more favorable than the 1,2,3-triazole linkage of the bradykinin-derived pseudopeptide 2a to preserve an effective affinity towards CatK, its protease target. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  14. Monitoring pancreatic carcinogenesis by the molecular imaging of cathepsin E in vivo using confocal laser endomicroscopy.

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    Hui Li

    Full Text Available The monitoring of pancreatic ductal adenocarcinoma (PDAC in high-risk populations is essential. Cathepsin E (CTSE is specifically and highly expressed in PDAC and pancreatic intraepithelial neoplasias (PanINs, and its expression gradually increases along with disease progression. In this study, we first established an in situ 7,12-dimethyl-1,2-benzanthracene (DMBA-induced rat model for PanINs and PDAC and then confirmed that tumorigenesis properties in this model were consistent with those of human PDAC in that CTSE expression gradually increased with tumor development using histology and immunohistochemistry. Then, using in vivo imaging of heterotopically implanted tumors generated from CTSE- overexpressing cells (PANC-1-CTSE in nude mice and in vitro imaging of PanINs and PDAC in DMBA-induced rats, the specificity of the synthesized CTSE-activatable probe was verified. Quantitative determination identified that the fluorescence signal ratio of pancreatic tumor to normal pancreas gradually increased in association with progressive pathological grades, with the exception of no significant difference between PanIN-II and PanIN-III grades. Finally, we monitored pancreatic carcinogenesis in vivo using confocal laser endomicroscopy (CLE in combination with the CTSE-activatable probe. A prospective double-blind control study was performed to evaluate the accuracy of this method in diagnosing PDAC and PanINs of all grades (>82.7%. This allowed us to establish effective diagnostic criteria for CLE in PDAC and PanINs to facilitate the monitoring of PDAC in high-risk populations.

  15. Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

    Science.gov (United States)

    Adamo, Michael; Sun, Guoyong; Qiu, Difei; Valente, Joseph; Lan, Wenkui; Song, Hangtian; Bolgar, Mark; Katiyar, Amit; Krishnamurthy, Girija

    2017-01-20

    Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination.

    Science.gov (United States)

    Wesołowska, Agnieszka; Basałaj, Katarzyna; Norbury, Luke J; Sielicka, Alicja; Wędrychowicz, Halina; Zawistowska-Deniziak, Anna

    2018-01-30

    No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

    DEFF Research Database (Denmark)

    Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel

    2012-01-01

    Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function...... as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified...

  18. Exploration of peptides that fit into the thermally vibrating active site of cathepsin K protease by alternating artificial intelligence and molecular simulation

    Science.gov (United States)

    Nishiyama, Katsuhiko

    2017-08-01

    Eighteen tripeptides that fit into the thermally vibrating active site of cathepsin K were discovered by alternating artificial intelligence and molecular simulation. The 18 tripeptides fit the active site better than the cysteine protease inhibitor E64, and a better inhibitor of cathepsin K could be designed considering these tripeptides. Among the 18 tripeptides, Phe-Arg-Asp and Tyr-Arg-Asp fit the active site the best and their structural similarity should be considered in the design process. Interesting factors emerged from the structure of the decision tree, and its structural information will guide exploration of potential inhibitor molecules for proteases.

  19. Heterologous expression, purification and characterization of human β-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

    Science.gov (United States)

    Miyazaki, Takatsugu; Kato, Tatsuya; Park, Enoch Y

    2018-02-03

    β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Disruption of Bombyx mori nucleopolyhedrovirus ORF71 (Bm71) results in inefficient budded virus production and decreased virulence in host larvae.

    Science.gov (United States)

    Zhang, Min-Juan; Cheng, Ruo-Lin; Lou, Yi-Han; Ye, Wan-Lu; Zhang, Tao; Fan, Xiao-Ying; Fan, Hai-Wei; Zhang, Chuan-Xi

    2012-08-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that selectively infects domestic silkworm. BmNPV ORF71 (Bm71) is not a core set gene in baculovirus and shares 92 % amino acid sequence identity with Autographa californica multinucleocapsid NPV ORF88 (Ac88/cg30). Previously, it has been reported that virus lacking Ac88 had no striking phenotypes in cell lines or host larvae. However, the exact role of Bm71 during BmNPV life cycle remains unknown. In the present study, we constructed a Bm71-disrupted (Bm71-D) virus and assessed the effect of the Bm71 disruption on viral replication and viral phenotype throughout the viral life cycle. Results showed that the Bm71-D bacmid could successfully transfect Bm5 cell lines and produce infectious budded virus (BV). But the BV titer was 10- to 100-fold lower than that of the wild-type (WT) virus during infection, and the decreased BV titer was rescued by Bm71 gene repair virus (Bm71-R). A larval bioassay showed that Bm71-D virus took 7.5 h longer than the WT to kill Bombyx mori larvae. Transmission electron microscopy analysis indicated that the Bm71-D virus-infected cells had typical virogenic stroma, bundles of nucleocapsids and polyhedra. Taken together, these results suggest that Bm71 has important implications for determining BV yield and virulence in viral life cycle even though it is not an essential gene for replication of BmNPV.

  1. The differential expression of BmGlcNAcase2 in strains of Bombyx mori (Lepidoptera: Bombycidae) with different susceptibility to Bombyx mori (Lepidoptera: Bombycidae) nucleopolyhedrovirus infection.

    Science.gov (United States)

    Hao, Zhu; Quanbing, Ma; Xiaoyong, Liu

    2015-01-01

    GlcNAcase is a glycosyl hydrolase located in the lysosomes of numerous organisms. Levels of the protein, β-N-acetylglucosaminidase 2 (GlcNAcase2), which is a member of the GlcNAcase family, are different in two strains of the silkworm Bombyx mori that have different resistance to Bombyx mori nucleopolyhedroviruses (BmNPVs). We identified six single-nucleotide differences in the GlcNAcase2 coding sequence between the 306 and NB strains. Five are silent changes, but one is a nonsynonymous mutation. Reverse transcription-polymerase chain reaction analysis showed that GlcNAcase2 mRNA levels in the NB strain were nearly 2.57 times higher compared with those in the 306 strain. In addition, GlcNAcase2 enzyme activity was much higher in the NB strain compared with that in the 306 strain. Together, these results indicate that GlcNAcase2 may be involved in variable BmNPV resistance in B. mori. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

  2. Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

    Science.gov (United States)

    Chen, Shuqing; Hou, Chengxiang; Bi, Honglun; Wang, Yueqiang; Xu, Jun; Li, Muwang; James, Anthony A; Huang, Yongping; Tan, Anjiang

    2017-04-15

    We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV immediate early-1 ( ie-1 ) and me53 genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy. IMPORTANCE Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species. Copyright © 2017 Chen et al.

  3. Differential expression of Cathepsin S and X in the spinal cord of a rat neuropathic pain model

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    Schmitz Beate

    2008-08-01

    Full Text Available Abstract Background Ample evidence suggests a substantial contribution of cellular and molecular changes in the spinal cord to the induction and persistence of chronic neuropathic pain conditions. While for a long time, proteases were mainly considered as protein degrading enzymes, they are now receiving growing interest as signalling molecules in the pain pathology. In the present study we focused on two cathepsins, CATS and CATX, and studied their spatiotemporal expression and activity during the development and progression of neuropathic pain in the CNS of the rat 5th lumbar spinal nerve transection model (L5T. Results Immediately after the lesion, both cathepsins, CATS and CATX, were upregulated in the spinal cord. Moreover, we succeeded in measuring the activity of CATX, which was substantially increased after L5T. The differential expression of these proteins exhibited the same spatial distribution and temporal progression in the spinal cord, progressing up to the medulla oblongata in the late phase of chronic pain. The cellular distribution of CATS and CATX was, however, considerably different. Conclusion The cellular distribution and the spatio-temporal development of the altered expression of CATS and CATX suggest that these proteins are important players in the spinal mechanisms involved in chronic pain induction and maintenance.

  4. Neutrophils Release Metalloproteinases during Adhesion in the Presence of Insulin, but Cathepsin G in the Presence of Glucagon

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    Natalia V. Fedorova

    2018-01-01

    Full Text Available In patients with reperfusion after ischemia and early development of diabetes, neutrophils can attach to blood vessel walls and release their aggressive bactericide agents, which damage the vascular walls. Insulin and 17β-estradiol (E2 relieve the vascular complications observed in metabolic disorders. In contrast, glucagon plays an essential role in the pathophysiology of diabetes. We studied the effect of hormones on neutrophil secretion during adhesion to fibronectin. Amino acid analysis revealed that proteins secreted by neutrophils are characterized by a stable amino acid profile enriched with glutamate, leucine, lysine, and arginine. The total amount of secreted proteins defined as the sum of detected amino acids was increased in the presence of insulin and reduced in the presence of glucagon. E2 did not affect the amount of protein secretion. Proteome analysis showed that in the presence of insulin and E2, neutrophils secreted metalloproteinases MMP-9 and MMP-8 playing a key role in modulation of the extracellular matrix. In contrast, glucagon induced the secretion of cathepsin G, a key bactericide protease of neutrophils. Cathepsin G can promote the development of vascular complications because of its proinflammatory activity and ability to stimulate neutrophil adhesion via the proteolysis of surface receptors.

  5. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    International Nuclear Information System (INIS)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid; Gjerloev, Simon; Birk, Jesper; Roepke, Carsten; Norrild, Bodil

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling

  6. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans.

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    Mark T Miedel

    Full Text Available Endoplasmic-reticulum associated degradation (ERAD is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY* was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.

  7. Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

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    Suman Kumar Nandy

    Full Text Available Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

  8. Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

    Science.gov (United States)

    Kelli Hoover; Merideth A. Humphries; Alyssa R. Genfron; James M. Slavicek

    2010-01-01

    Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion...

  9. Relation of bone mineral density with homocysteine and cathepsin K levels in postmenopausal women

    Directory of Open Access Journals (Sweden)

    Madhukar Mittal

    2018-01-01

    Full Text Available Background: Homocysteine (HCY interferes with collagen cross-linking in bones and stimulates osteoclast activity. The activated osteoclasts secrete cathepsin K (CathK, a cysteine protease, in eminent quantity during bone resorption. Hyperhomocysteinemia may effect bone mineral density (BMD through CathK. We, therefore, examined the relation between HCY and BMD along with CathK, 25-hydroxyvit-D (25[OH]D, intact parathyroid hormone (iPTH, and Vitamin B12. Materials and Methods: We recruited a total of 93 postmenopausal women between the age group of 45–60 years, attending the Endocrinology outpatient department at King George's Medical University, Lucknow. BMD was done by DXA scan using Hologic QDR1000 system. Based on the WHO criteria, patients were segregated into three groups as follows; normal bone mass, osteopenia, and osteoporosis. All women underwent routine biochemical laboratory parameters, HCY, Vitamin B12, and CathK levels. Results: Among 93 postmenopausal women, 56% (52 had osteoporosis. Nineteen percent (18 had normal BMD (mean age, 53.22 ± 8.5 years and 23 (25% had osteopenia (mean age 52.86 ± 6.67 years. The mean age in the osteoporetic group was 56.2 ± 6.9 years. The median (interquartile range levels of HCY in the three groups were 14.5 μmol/L (12.2–24.7, 15.05 μmol/L (12.1–19.9 and 13.2 μmol/L (10.3–17.0, respectively. CathK levels were similar in three groups 7.6 ng/ml (7.0–80.5, 8.3 ng/ml (7.3–8.5, and 8.6 ng/ml (7.2–8.9. Both HCY and CathK were found positively associated with serum phosphorus (r = 0.584, P < 2.01 and r = 0.249, P < 0.05, respectively. Levels of HCY positively correlate with PTH (r = 0.303, P < 0.01 and inversely with Vitamin B12 (r = −0.248, P < 0.05. No significant association was seen between CathK level and 25(OH D, iPTH, serum calcium. Conclusion: Low bone mass by DXA is a significant problem in postmenopausal females. HCY and CathK do not reliably correlate with bone loss in

  10. Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

    Science.gov (United States)

    Anuracpreeda, Panat; Srirakam, Thippawan; Pandonlan, Sudarat; Changklungmoa, Narin; Chotwiwatthanakun, Charoonroj; Tinikul, Yotsawan; Poljaroen, Jaruwan; Meemon, Krai; Sobhon, Prasert

    2014-08-01

    Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. The selective cathepsin K inhibitor MIV-711 attenuates joint pathology in experimental animal models of osteoarthritis.

    Science.gov (United States)

    Lindström, Erik; Rizoska, Biljana; Tunblad, Karin; Edenius, Charlotte; Bendele, Alison M; Maul, Don; Larson, Michael; Shah, Neha; Yoder Otto, Valerie; Jerome, Chris; Grabowska, Urszula

    2018-03-09

    MIV-711 is a highly potent and selective cathepsin K inhibitor. The current article summarizes the therapeutic effects of MIV-711 on joint pathology in rabbits subjected to anterior cruciate ligament transection (ACLT), and the prophylactic effects on joint pathology in dogs subjected to partial medial meniscectomy, two surgical models of osteoarthritis (OA). Starting 1 week after surgery, rabbits were dosed daily via oral gavage with either MIV-711 or vehicle (n = 7/group) for 7 weeks. The four treatment groups were: (1) sham + vehicle; (2) ACLT + vehicle; (3) ACLT + MIV-711, 30 µmol/kg and (4) ACLT + MIV-711, 100 µmol/kg. Subchondral bone and articular cartilage structures were assessed by µCT, histomorphometry, and scoring. Dogs subjected to partial medial meniscectomy received either MIV-711 (30 µmol/kg) or vehicle (n = 15/group) via oral gavage once daily, starting 1 day before meniscectomy, for 28 days. Cartilage degradation was assessed at the macroscopic and microscopic levels. The exposures of MIV-711 were assessed in both studies and biomarkers reflecting bone resorption (HP-1 in rabbits, CTX-I in dogs) and cartilage degradation (CTX-II) were measured. In ACLT rabbits, MIV-711 decreased HP-1 levels by up to 72% (p subchondral bone plate and reduced trabecular bone volume in the femur and tibia. These effects were reversed by MIV-711. ACLT resulted in cartilage thickening, which was attenuated by MIV-711. MIV-711 did not affect osteophyte formation or Mankin scores. In dogs, MIV-711 reduced CTX-I and CTX-II levels by 86% (p subchondral bone loss and partially attenuates cartilage pathology in two animal models of OA. These beneficial effects of MIV-711 on joint pathology are observed in conjunction with decreases in bone and cartilage biomarkers that have been shown to be clinically attainable in human. The data support the further development of MIV-711 for the treatment of OA.

  12. Downregulation of cathepsin G reduces the activation of CD4+ T cells in murine autoimmune diabetes.

    Science.gov (United States)

    Zou, Fang; Lai, Xiaoyang; Li, Jing; Lei, Shuihong; Hu, Lei

    2017-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease due to progressive injury of islet cells mediated by T lymphocytes (T cells). Our previous studies have shown that only cathepsin G (CatG), not other proteases, is involved in the antigen presentation of proinsulin, and if the presentation is inhibited, the activation of CD4+ T cells induced by proinsulin is alleviated in T1DM patients, and CatG-specific inhibitor reduces the activation of CD4+ cells induced by proinsulin in T1DM patients. Therefore, we hypothesize that CatG may play an important role in the activation of CD4+ T cells in T1DM. To this end, mouse studies were conducted to demonstrate that CatG impacts the activation of CD4+ T cells in non-obese diabetic (NOD) mice. CatG gene expression and the activation of CD4+ T cells were examined in NOD mice. The effect of CatG inhibitor was investigated in NOD mice on the activation of CD4+ T cells, islet β cell function, islet inflammation and β-cell apoptosis. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on the activation status of CD4+ T cells and the progression of diabetes. During the pathogenesis of diabetes, the expression level of CatG in NOD mice gradually increased and the CD4+ T cells were gradually activated, resulting in more TH1 cells and less TH2 and Treg cells. Treatment with CatG-specific inhibitor reduced the blood glucose level, improved the function of islet β cells and reduced the activation of CD4+ T cells. Early application of CatG siRNA improved the function of islet β cells, reduced islet inflammation and β cell apoptosis, and lowered the activation level of CD4+ T cells, thus slowing down the progression of diabetes.

  13. β-Endorphin biotransformation in brain: Formation of γ-endorphin by a synaptosomal plasma membrane associated endopeptidase distinct from cathepsin D

    NARCIS (Netherlands)

    Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de

    1980-01-01

    cSPM preparations of rat brain contain a peptidase activity which generates γ-endorphin from β-endorphin. Some properties of this enzyme were studied and compared with those of cathepsin D. Maximal accumulation of γ-endorphin upon digestion of β-endorphin with a cSPM preparation was found at neutral

  14. Activation of cathepsin L contributes to the irreversible depolarization induced by oxygen and glucose deprivation in rat hippocampal CA1 neurons.

    Science.gov (United States)

    Kikuta, Shogo; Murai, Yoshinaka; Tanaka, Eiichiro

    2017-01-01

    Oxygen and glucose deprivation (OGD) elicits a rapid and irreversible depolarization with a latency of ∼5min in intracellular recordings of hippocampal CA1 neurons in rat slice preparations. In the present study, we examined the role of cathepsin L in the OGD-induced depolarization. OGD-induced depolarizations were irreversible as no recovery of membrane potential was observed. The membrane potential reached 0mV when oxygen and glucose were reintroduced immediately after the onset of the OGD-induced rapid depolarization. The OGD-induced depolarizations became reversible when the slice preparations were pre-incubated with cathepsin L inhibitors (types I and IV at 0.3-2nM and 0.3-30nM, respectively). Moreover, pre-incubation with these cathepsin inhibitors prevented the morphological changes, including swelling of the cell soma and fragmentation of dendrites, observed in control neurons after OGD. These findings suggest that the activation of cathepsin L contributes to the irreversible depolarization produced by OGD. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. 4-tert-Octylphenol stimulates the expression of cathepsins in human breast cancer cells and xenografted breast tumors of a mouse model via an estrogen receptor-mediated signaling pathway

    International Nuclear Information System (INIS)

    Lee, Hye-Rim; Choi, Kyung-Chul

    2013-01-01

    Highlights: ► Cathepsins B and D were markedly enhanced by octylphenol (OP) in MCF-7 cells. ► OP may accelerate breast cancer cell growth and cathepsins via ER-mediated signaling. ► Breast cancer cells exposed with OP to mouse model were more aggressive. ► OP can promote metastasis through the amplification of cathepsins B and D via ER-mediated signaling pathway. -- Abstract: Endocrine disrupting chemicals (EDCs) are defined as environmental compounds that modulate steroid hormone receptor-dependent responses an abnormal manner, resulting in adverse health problems for humans such as cancer growth and metastasis. Cathepsins are proteases that have been implicated in cancer progression. However, there have been few studies about the association between cathepsins and estrogenic chemicals during the cancer progression. In this study, we examined the effect(s) of 4-tert-octylphenol (OP), a potent EDC, on the expression of cathepsins B and D in human MCF-7 breast cancer cells and a xenograft mouse model. Treatment with OP significantly induced the proliferation MCF-7 cells in an MTT assay. In addition, the expression of cathepsins B and D was markedly enhanced in MCF-7 cells at both the transcriptional and the translational levels following treatment with E2 or OP up to 48 h. These results demonstrated the ability of OP to disrupt normal transcriptional regulation of cathepsins B and D in human breast cancer cells. However, the effects of OP on cell growth or overexpression of cathepsins by inhibiting ER-mediated signaling were abolished by an ER antagonist and siRNA specific for ERα. In conclusion, our findings suggest that OP at 10 −6 M, like E2, may accelerate breast cancer cell proliferation and the expression of cathepsins through an ER-mediated signaling pathway. In addition, the breast cancer cells exposed with OP to a xenograft mouse model were more aggressive according to our histological analysis and showed markedly increased expression of

  16. Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Taneo, Jun; Adachi, Takumi [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Yoshida, Aiko; Takayasu, Kunio [Responses to Environmental Signals and Stresses, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto, Kyoto 606-8501 (Japan); Takahara, Kazuhiko, E-mail: ktakahar@zoo.zool.kyoto-u.ac.jp [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan); Inaba, Kayo [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan)

    2015-03-13

    Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.

  17. Increased Plasma Cathepsin S at the Time of Percutaneous Transluminal Angioplasty is Associated with 6-Months’ Restenosis of the Femoropopliteal Artery

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    Mijovski Mojca Bozic

    2018-01-01

    Full Text Available Background: We tested the hypothesis that increased levels of cathepsin S and decreased levels of cystatin C in plasma at the time of percutaneous transluminal angioplasty (PTA are associated with the occurrence of 6-months’ restenosis of the femoropopliteal artery (FPA. Methods: 20 patients with restenosis and 24 matched patients with patent FPA after a 6-months follow-up were in - cluded in this study. They all exhibited disabling claudication or critical limb ischemia and had undergone technically successful PTA. They were all receiving statins and ACE in hi - bitors (or angiotensin II receptor antagonist before the PTA and the therapy did not change throughout the observational period. Plasma concentrations of C-reactive protein were < 10 mg/L and of creatinine within the reference range at the time of the PTA. Plasma concentration and activity of cathepsin S, together with its potent inhibitor cystatin C, were measured the day before and the day after the PTA. Results: The increased plasma concentration and activity of cathepsin S at the time of PTA was associated with the occurrence of 6-months’ restenosis of FPA, independently of established risk factors (lesion complexity, infrapopliteal run-off vessels, type of PTA, age, gender, smoking, diabetes, lipids and of cystatin C. Plasma cystatin C concentration was not associated with restenosis and did not correlate with cathepsin S activity and concentration in the plasma. Conclusion: Increased level of plasma cathepsin S at the time of PTA is associated with 6-months’ restenosis of PTA, independently of established risk factors.

  18. Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

    International Nuclear Information System (INIS)

    Berretta, Marcelo F.; Deshpande, Mandar; Crouch, Erin A.; Passarelli, A. Lorena

    2006-01-01

    We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins

  19. Quantitative and ultrastructural changes in the haemocytes of Spodoptera littoralis (Boisd.) treated individually or in combination with Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) and azadirachtin.

    Science.gov (United States)

    Shaurub, El-Sayed H; Abd El-Meguid, Afaf; Abd El-Aziz, Nahla M

    2014-10-01

    The total haemocyte count (THC) and the possible ultrastructural alterations induced in the haemocytes of the fourth larval instars of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), 96 h post-feeding on a semi-synthetic diet, treated with the LC50 of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) and the LC50 of azadirachtin alone, and the LC25 of SpliMNPV combined with the LC25 of azadirachtin were studied and compared to the control. Single treatment with the virus and azadirachtin or combined treatment significantly decreased the THC compared to the control. There are five types of haemocytes in S. littoralis: prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. The most common symptoms in granulocytes and plasmatocytes, the main affected cell types, due to viral infection were the presence of virogenic stroma, peripheral dispersion of the chromatin and disappearance of the nucleoli. However, the most common symptoms in these two types of haemocytes due to treatment with azadirachtin were the presence of rough endoplasmic reticulum filled with fibrous materials, due to probably apoptosis, in their cisternae and disorganization of mitochondria (looped, vacuolated and swollen). In addition, the cytoplasm of granulocytes was vacuolated with the appearance of autophagic lysosomes, while plasmatocytes showed ruptured cell membrane and folded nuclear envelope. Combined treatment with the NPV and azadirachtin induced the same pathological changes which were recorded from individual treatment with the virus or azadirachtin to the same haemocytes. It can be concluded that the change in the THC and ultrastructure of granulocytes and plasmatocytes may affect the cellular-mediated immune response in S. littoralis. Moreover, it seems likely that mitochondria were the target site of azadirachtin, as they were affected in both granulocytes and plasmatocytes treated with azadirachtin alone or in

  20. Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus

    Directory of Open Access Journals (Sweden)

    Chie Goto

    2015-03-01

    Full Text Available Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV, and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%. In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.

  1. Sildenafil Decreases BACE1 and Cathepsin B Levels and Reduces APP Amyloidogenic Processing in the SAMP8 Mouse.

    Science.gov (United States)

    Orejana, Lourdes; Barros-Miñones, Lucía; Jordan, Joaquin; Cedazo-Minguez, Angel; Tordera, Rosa M; Aguirre, Norberto; Puerta, Elena

    2015-06-01

    The senescence-accelerated mouse-prone 8 (SAMP8), used as a model of aging, displays many established pathological features of Alzheimer's disease. Cognitive impairments and increased levels of hyperphosphorylated tau are found in the hippocampus of SAMP8 mice along with an increased β-secretase activity and amyloid-β (Aβ) depositions that increase in number and extent with age. Based on a previous study from our laboratory showing an amelioration of cognitive impairments and tau pathology by sildenafil, in this study we tested whether this drug could also modulate the amyloid precursor protein amyloidogenic processing in this mouse model. Our results show that the protein levels of the β-secretases β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B are higher in the hippocampus of 9-month-old SAMP8 mice than those of age-matched senescence-resistant-1. Sildenafil (7.5mg/kg for 4 weeks) attenuated learning and memory impairments shown by SAMP8 mice in the passive avoidance test. The increased expression of β-site amyloid precursor protein cleaving enzyme 1 was also reduced by sildenafil, an effect paralleled to decreases in the activities of two β-site amyloid precursor protein cleaving enzyme 1 modulators, calpain and cyclin-dependent kinase 5 protein. Interestingly, sildenafil enhanced both Akt and glycogen synthase kinase-3β (ser9) phosphorylation, which could be mediating the reduction in cathepsin B levels found in the hippocampus of sildenafil-treated SAMP8 mice. Sildenafil-induced reduction in β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B expression in SAMP8 mice was associated with a decrease in hippocampal Aβ42 levels which, in turn, could mediate the parallel decline in glial fibrillary acidic protein expression observed in these animals. These findings highlight the therapeutic potential of sildenafil in Alzheimer's disease pathogenesis. © The Author 2014. Published by Oxford University Press on behalf of

  2. Single nucleotide polymorphisms of cathepsin S and the risks of asthma attack induced by acaroid mites.

    Science.gov (United States)

    Li, Chaopin; Chen, Qi; Jiang, Yuxin; Liu, Zhiming

    2015-01-01

    To investigate association between the three single nucleotide polymorphisms (SNPs, rs146456111, rs143154304 and rs147260142) in cathepsin S (Cat S) and the risks of allergic asthma attack induced by the acaroid mites in the Chinese population. A case-control study was performed in 412 cases and 454 volunteers/controls to evaluate the effects of three SNPs in Cat S on the risks of asthma attack. The genotypes were determined using polymerase chain reaction (PCR) and cleaved amplification polymorphism sequence-tagged sites (PCR-RFLP). The frequencies of genotypes and alleles in these SNPs in the asthmatic group were also analyzed between the two groups. The locus of rs146456111 in Cat S gene, the allele frequency of A and C in asthmatic group were significantly different from the control group (χ(2) = 184.425, P = 0.000), and the difference was significant regarding the distribution of the genotypes (AA, AC, and CC) between asthmatic subjects and normal controls (χ(2) = 177.915, P = 0.000). Logistic regression analysis revealed that the AC, CC, and AC + CC genotypes were significantly increased with the risk of asthma (AC vs. AA, OR = 4.013, 95% CI = 2.989-4.751, P = 0.000; CC vs. AA, OR = 3.167, 95% CI = 2.483-3.785, P = 0.000; AC + CC vs. AA, OR = 3.418, 95% CI = 2.381-4.214, P = 0.000, respectively), compared with AA genotype. Moreover, by comparison with allele A, allele C (OR = 2.187, 95% CI = 1.743-2.281, P asthma; For the locus of rs143154304, compared with the allele frequency G with A in control group, there was no difference (χ(2) = 1.434, P = 0.231) in that of asthmatic group, as well as the distributions of the genotypes (AA, AG, and GG) between asthmatic subjects and normal controls (χ(2) = 1.997, P = 0.369); Logistic regression analysis showed that the AG, GG, and AG + GG genotypes were no risk to asthma (AG vs. AA, OR = 0.991, 95% CI = 0.625-1.507, P = 0.968; GG vs. AA, OR = 0.812, 95% CI = 0.525-1.258, P = 0.352; AG + GG vs. AA, OR = 0.914, 95

  3. Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

    Science.gov (United States)

    Giroud, Maude; Dietzel, Uwe; Anselm, Lilli; Banner, David; Kuglstatter, Andreas; Benz, Jörg; Blanc, Jean-Baptiste; Gaufreteau, Delphine; Liu, Haixia; Lin, Xianfeng; Stich, August; Kuhn, Bernd; Schuler, Franz; Kaiser, Marcel; Brun, Reto; Schirmeister, Tanja; Kisker, Caroline; Diederich, François; Haap, Wolfgang

    2018-04-26

    Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( K i < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC 50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

  4. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation......, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge...

  5. Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Chida, Kohsuke; Taguchi, Meiko

    2011-01-01

    Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes

  6. Induction of premalignant host responses by cathepsin x/z-deficiency in Helicobacter pylori-infected mice.

    Directory of Open Access Journals (Sweden)

    Sabine Krueger

    Full Text Available Helicobacter pylori are responsible for the induction of chronic gastric inflammation progressing to atrophy, metaplasia, and gastric cancer. The overexpression of Cathepsin X/Z (Ctsz in H. pylori-infected mucosa and gastric cancer is mediated predominantly by an augmented migration of ctsz(-/-positive macrophages and the up-regulation of Ctsz in tumor epithelium. To explore the Ctsz-function in the context of chronic inflammation and the development of preneoplastic lesions, we used Ctsz-deficient mice in a H. pylori gastritis model. Ctsz (-/- and wild-type (wt mice were infected with H. pylori strain SS1. The mice were sacrificed at 24, 36, and 50 weeks post infection (wpi. The stomach was removed, and gastric strips were snap-frozen or embedded and stained with H&E. Tissue sections were scored for epithelial lesions and inflammation. Ki-67 and F4/80 immunostaining were used to measure epithelial cell proliferation and macrophage infiltration, respectively. The upregulation of compensating cathepsins and cytokines were confirmed by Western blotting and quantitative RT-PCR. SS1-infected wt and ctsz (-/- mice showed strong inflammation, foveolar hyperplasia, atrophy, and cystically-dilated glands. However, at 50 wpi, ctsz (-/- mice developed significantly more severe spasmolytic polypeptide-expressing metaplasia (SPEM, showed enhanced epithelial proliferation, and higher levels of infiltrating macrophages. Induction of cytokines was higher and significantly prolonged in ctsz (-/- mice compared to wt. Ctsz deficiency supports H. pylori-dependent development of chronic gastritis up to metaplasia, indicating a protective, but not proteolytic, function of Ctsz in inflammatory gastric disease.

  7. Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise among limits of detection, reduced time, and resources.

    Science.gov (United States)

    Dumas, Jerald E; Platt, Manu O

    2013-07-01

    Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.

  8. Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise between limits of detection, reduced time, and resources

    Science.gov (United States)

    Dumas, Jerald E.; Platt, Manu O.

    2013-01-01

    Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as cleared bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 hours to 10 minutes, incubation assay time from overnight to 4 hours, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings. PMID:23532386

  9. Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas

    International Nuclear Information System (INIS)

    Hasui, Kazuhisa; Wang, Jia; Jia, Xinshan; Tanaka, Masashi; Nagai, Taku; Matsuyama, Takami; Eizuru, Yoshito

    2011-01-01

    This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV + lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV + NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL

  10. Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions

    DEFF Research Database (Denmark)

    Poulsen, Christian Bo; Al-Mashhadi, Ahmed Ludvigsen; von Wachenfeldt, Karin

    2016-01-01

    and results Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12 weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n = 9) was performed before inclusion and after 3 months of treatment. Blood samples....... There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p = 0.......03) with no difference in CD68 or CD163 positivity. Conclusions In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal....

  11. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K

    International Nuclear Information System (INIS)

    Sousa, Lorena Ramos Freitas de; Silva, Jame's A. da; Vieira, Paulo Cezar; Costa, Maisa Borges; Santos, Mirley Luciene dos; Menezes, Antonio Carlos Severo; Sbardelotto, Aline Borba; Pessoa, Claudia do O; Moraes, Manoel Odorico de

    2014-01-01

    A new flavonoid, catechin-3-O-(3 - O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3 - O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC 50 = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC 50 = 36.80 μM and IC 50 = 25.37 μM, respectively (author)

  12. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

  13. Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingyun, E-mail: lingyunlee@126.com [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Gao, Luyan [Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Song, Yunzhen; Qin, Zheng-Hong [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Liang, Zhongqin, E-mail: liangzhongqin@suda.edu.cn [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China)

    2016-02-12

    Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.

  14. Effect of mefenamic acid on the immunity and hemostatic system of cancer patients and on the activity of cathepsin D-like protease in colonic cancer tissue

    International Nuclear Information System (INIS)

    Klyachkin, B.M.; Basargin, S.T.; Timofeev, I.V.; Khaliulin, Yu.G.; Dorofeev, S.A.; Alekseenko, L.D.; Gumenyuk, M.L.

    1992-01-01

    The study of the effect of sodium mefenaminate on radiation resistance of mice yielded positive results. Clinical investigations showed mefenamic acid to decrease the activity of cathepsin D-like protease in colonic cancer tissue. The acid field to affect the proteolytic activity of the normal mucosa. It revealed an immunomodulating activity and influenced the hemostatic system which usually manifested itself in amelioration of hypercoagulation

  15. Mapping the Pro-Peptide of the Schistosoma mansoni Cathepsin B1 Drug Target: Modulation of Inhibition by Heparin and Design of Mimetic Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Jílková, Adéla; Vondrášek, Jiří; Marešová, Lucie; Caffrey, C. R.; Mareš, Michael

    2011-01-01

    Roč. 6, č. 6 (2011), s. 609-617 ISSN 1554-8929 R&D Projects: GA ČR GA203/09/1585; GA AV ČR KJB400550516; GA AV ČR IAA400550705 Grant - others:NATO(XE) NATO LST/CLG 980187 Institutional research plan: CEZ:AV0Z40550506 Keywords : Schistosoma mansoni * cathepsin B * propeptide Subject RIV: CE - Biochemistry Impact factor: 6.446, year: 2011

  16. Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cavailles, V; Augereau, P; Garcia, M; Rochefort, H

    1988-03-25

    The estrogen-induced 52K protein secreted by human breast cancer cells is a lysosomal protease recently identified as a pro-cathepsin D by sequencing several cDNA clones isolated from MCF/sub 7/ cells. Using one of these clones, the authors detected, in MCF/sub 7/ cells a 2.2 kb mRNA whose level was rapidly increased 4- to 10-fold by estradiol, but not by other classes of steroids. Other mitogens, such as epidermal growth factor and insulin, also induced the 2.2 kb mRNA in a dose-dependent manner. Induction with epidermal growth factor was as rapid but was 2- to 3-fold lower than with estradiol. Antiestrogens had no effect on the 52K-cathepsin-D mRNA in MCF/sub 7/ cells, but became estrogen agonists in two antiestrogen-resistant sublines R/sub 27/ and LY2. The use of transcription and translation inhibitors and nuclear run-on experiments indicate that estradiol enhances transcription of the 52K-cathepsin-D gene in MCF/sub 7/ cells.

  17. The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

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    Violeta Morin

    Full Text Available Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

  18. Azilsartan increases levels of IL-10, down-regulates MMP-2, MMP-9, RANKL/RANK, Cathepsin K and up-regulates OPG in an experimental periodontitis model.

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    Aurigena Antunes de Araújo

    Full Text Available AIMS: The aim of this study was to evaluate the effects of azilsartan (AZT on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs, receptor activator of nuclear factor κB ligand (RANKL, receptor activator of nuclear factor κB (RANK, osteoprotegerin (OPG, cyclooxygenase-2 (COX-2, and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into 5 groups of 10 rats each: (1 nonligated, water; (2 ligated, water; (3 ligated, 1 mg/kg AZT; (4 ligated, 5 mg/kg AZT; and (5 ligated, 10 mg/kg AZT. All groups were treated with saline or AZT for 10 days. Periodontal tissues were analyzed by histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1β, IL-10, TNF-α, myeloperoxidase (MPO, and glutathione (GSH were determined by ELISA. RESULTS: Treatment with 5 mg/kg AZT resulted in reduced MPO (p<0.05 and IL-1β (p<0.05, increased levels of IL-10 (p<0.05, and reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and increased expression of OPG. CONCLUSIONS: These findings reveal that AZT increases anti-inflammatory cytokines and GSH and decreases bone loss in ligature-induced periodontitis in rats.

  19. Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

    Science.gov (United States)

    Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

    2012-02-10

    Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

    Science.gov (United States)

    Kwon, Chang Woo; Yang, Hee; Yeo, SuBin; Park, Kyung-Min; Jeong, Ae Jin; Lee, Ki Won; Ye, Sang-Kyu; Chang, Pahn-Shick

    2018-12-01

    Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar K i value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).

  1. Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

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    Jian Wang

    Full Text Available The aim of this study was to characterize the oncogenic function and mechanism of Cathepsin Z (CTSZ at 20q13.3, a frequently amplified region in hepatocellular carcinoma (HCC. Real-time PCR were used to compare CTSZ expression between paired HCC tumor and non-tumor specimens. CTSZ gene was stably transfected into HCC line QGY-7703 cells and its role in tumorigenicity and cell motility was characterized by soft agar, wound-healing, transwell invasion and cell adhesion assay, and tumor xenograft mouse model. Western blot analysis was used to study expression of proteins associated with epithelial-mesenchymal transition (EMT.Upregulation of CTSZ was detected in 59/137 (43% of primary HCCs, which was significantly associated with advanced clinical stage (P = 0.000. Functional study found that CTSZ could increase colony formation in soft agar and promote cell motility. Further study found that the metastatic effect of CTSZ was associated with its role in inducing epithelial-mesenchymal transition (EMT by upregulating mesenchymal markers (fibronectin and vimentin and downregulating epithelial markers (E-cadherin and α-catenin. In addition, CTSZ could also upregulate proteins associated with extracellular matrix remodeling such as MMP2, MMP3 and MMP9. Taken together, our data suggested that CTSZ was a candidate oncogene within the 20q13 amplicon and it played an important role in HCC metastasis.

  2. Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

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    Kristine Porter

    Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  3. Molecular characterization and expression analysis of cathepsin C in Chinese giant salamander (Andrias davidianus after Aeromonas hydrophila infection

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    Zisheng Wang

    2018-03-01

    Full Text Available Background: Cathepsin C (CTSC (dipeptidyl peptidase I, DPPI, is a member of the papain superfamily of cysteine proteases and involves in a variety of host reactions. However, the information of CTST in Chinese giant salamander (Andrias davidianus, an amphibian species with important evolutionary position and economic values, remained unclear. Results: The full-length salamander CTSC cDNA contained a 96 bp of 5′-UTR, a 1392 bp of ORF encoding 463 amino acids, and a 95 bp of 3′-UTR. The salamander CTSC possessed several sequence features similar to other reported CTSCs such as a signal peptide, a propeptide and a mature peptide. The active site triad of Cys, His and Asn were also found existing in salamander CTSC. Salamander CTSC mRNA was constitutively expressed in all the examined tissues with significantly variant expression level. The highest expression of CTSC was in intestine, followed with stomach, spleen, lung and brain. Following Aeromonas hydrophila infection for 12 h, salamander CTSC was significantly up-regulated in several tissues including lung, spleen, brain, kidney, heart, stomach and skin. Conclusion: CTSC plays roles in the immune response to bacterial infection, which provided valuable information for further studying the functions of CTSC in salamander. Keywords: cDNA, CTSC, Dipeptidyl peptidase I, Gene expression, Hydrophila, Immune, Peptide, Sequence, Tissue

  4. THE PROGNOSIS SIGNIFICANCE OF CATHEPSIN-D EXPRESSION IN THE DIFFERENT LOCATIONS IN AXILLARY NODES NEGATIVE CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: The aim of this study was to investigate Cathepsin-D (Cath-D) expression in different location and its relationship with prognosis in the axillary lymph nodes negative (ANN) breast cancer patients. Methods: Cath-D expression in 192 cases of breast carcinoma were examined by immunohistochemistry. Depending on different parts of expression, three evaluating methods were used, compared and analysed. Results: The positive rate of Cath-D expression in ANN breast cancer with poor prognosis group and axillary nodes positive (ANP) group were significantly higher than that in ANN breast cancer with good prognosis group (x2=23.20, P0.05). Cath-D expression in stromal cells had no statistical difference among the three groups (x2=1.56, P>0.05). When the Cath-D expression in cancer and stromal cells were counted into the positive rate, it was near the same (u1=0.47, u2=1.41, P>0.05). Conclusion: These results suggest that Cath-D expression is one of the powerful prognostic markers in ANN breast cancer. It's a reliable, practical, and convenient method to observe and evaluate Cath-D expression in cancer cells.

  5. Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary.

    Science.gov (United States)

    Morais, Roberto D V S; Thomé, Ralph G; Santos, Hélio B; Bazzoli, Nilo; Rizzo, Elizete

    2016-04-01

    In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. The cathepsin B inhibitor z-FA-CMK induces cell death in leukemic T cells via oxidative stress.

    Science.gov (United States)

    Liow, K Y; Chow, Sek C

    2018-01-01

    The cathepsin B inhibitor benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was recently found to induce apoptosis at low concentrations in Jurkat T cells, while at higher concentrations, the cells die of necrosis. In the present study, we showed that z-FA-CMK readily depletes intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) generation. The toxicity of z-FA-CMK in Jurkat T cells was completely abrogated by N-acetylcysteine (NAC), suggesting that the toxicity mediated by z-FA-CMK is due to oxidative stress. We found that L-buthionine sulfoximine (BSO) which depletes intracellular GSH through the inhibition of GSH biosynthesis in Jurkat T cells did not promote ROS increase or induce cell death. However, NAC was still able to block z-FA-CMK toxicity in Jurkat T cells in the presence of BSO, indicating that the protective effect of NAC does not involve GSH biosynthesis. This is further corroborated by the protective effect of the non-metabolically active D-cysteine on z-FA-CMK toxicity. Furthermore, in BSO-treated cells, z-FA-CMK-induced ROS increased which remains unchanged, suggesting that the depletion of GSH and increase in ROS generation mediated by z-FA-CMK may be two separate events. Collectively, our results demonstrated that z-FA-CMK toxicity is mediated by oxidative stress through the increase in ROS generation.

  7. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer

    International Nuclear Information System (INIS)

    Herszényi, László; Farinati, Fabio; Cardin, Romilda; István, Gábor; Molnár, László D; Hritz, István; De Paoli, Massimo; Plebani, Mario; Tulassay, Zsolt

    2008-01-01

    Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging. The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%), CATB (82%), uPA (69%), CATL (41%) were higher than those of CEA or CA 19-9 (30% and 18%, respectively). PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%). High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004), CATL (P = 0.02), PAI-1 (P = 0.01) and CA 19-9 (P = 0.004) had a significant prognostic impact. PAI-1 (P = 0.001), CATB (P = 0.04) and CA 19-9 (P = 0.02) proved as independent prognostic variables. At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer

  8. Selective Cathepsin S Inhibition with MIV-247 Attenuates Mechanical Allodynia and Enhances the Antiallodynic Effects of Gabapentin and Pregabalin in a Mouse Model of Neuropathic Pain.

    Science.gov (United States)

    Hewitt, Ellen; Pitcher, Thomas; Rizoska, Biljana; Tunblad, Karin; Henderson, Ian; Sahlberg, Britt-Louise; Grabowska, Urszula; Classon, Björn; Edenius, Charlotte; Malcangio, Marzia; Lindström, Erik

    2016-09-01

    Cathepsin S inhibitors attenuate mechanical allodynia in preclinical neuropathic pain models. The current study evaluated the effects when combining the selective cathepsin S inhibitor MIV-247 with gabapentin or pregabalin in a mouse model of neuropathic pain. Mice were rendered neuropathic by partial sciatic nerve ligation. MIV-247, gabapentin, or pregabalin were administered alone or in combination via oral gavage. Mechanical allodynia was assessed using von Frey hairs. Neurobehavioral side effects were evaluated by assessing beam walking. MIV-247, gabapentin, and pregabalin concentrations in various tissues were measured. Oral administration of MIV-247 (100-200 µmol/kg) dose-dependently attenuated mechanical allodynia by up to approximately 50% reversal when given as a single dose or when given twice daily for 5 days. No behavioral deficits were observed at any dose of MIV-247 tested. Gabapentin (58-350 µmol/kg) and pregabalin (63-377 µmol/kg) also inhibited mechanical allodynia with virtually complete reversal at the highest doses tested. The minimum effective dose of MIV-247 (100 µmol/kg) in combination with the minimum effective dose of pregabalin (75 µmol/kg) or gabapentin (146 µmol/kg) resulted in enhanced antiallodynic efficacy without augmenting side effects. A subeffective dose of MIV-247 (50 µmol/kg) in combination with a subeffective dose of pregabalin (38 µmol/kg) or gabapentin (73 µmol/kg) also resulted in substantial efficacy. Plasma levels of MIV-247, gabapentin, and pregabalin were similar when given in combination as to when given alone. Cathepsin S inhibition with MIV-247 exerts significant antiallodynic efficacy alone, and also enhances the effect of gabapentin and pregabalin without increasing side effects or inducing pharmacokinetic interactions. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  9. Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

    Science.gov (United States)

    Aich, Pulakesh; Biswas, Sampa

    2018-06-01

    Pro-domain of a cysteine cathepsin contains a highly conserved Ex 2 Rx 2 Fx 2 Nx 3 Ix 3 N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

  10. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

    Science.gov (United States)

    Ferrara, Taíse Fernanda da Silva; Schneider, Vanessa Karine; Kishi, Luciano Takeshi; Carmona, Adriana Karaoglanovic; Alves, Marcio Fernando Madureira; Belasque-Júnior, Jose; Rosa, José César; Hunter, Wayne Brian; Henrique-Silva, Flávio; Soares-Costa, Andrea

    2015-01-01

    Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  11. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae: A Putative Target for Control of Citrus Huanglongbing.

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    Taíse Fernanda da Silva Ferrara

    Full Text Available Huanglonbing (HLB is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB. DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM. The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM and CaneCPI-4 (Ki = 0.05 nM and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM. RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  12. Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

    Directory of Open Access Journals (Sweden)

    Gao-Feng Qiu

    2009-01-01

    Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

  13. Profiling trait anxiety: transcriptome analysis reveals cathepsin B (Ctsb as a novel candidate gene for emotionality in mice.

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    Ludwig Czibere

    Full Text Available Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait "anxiety". We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB or low (LAB anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7, cathepsin B (Ctsb, muscleblind-like 1 (Mbnl1, metallothionein 1 (Mt1, solute carrier family 25 member 17 (Slc25a17, tribbles homolog 2 (Trib2, zinc finger protein 672 (Zfp672, syntaxin 3 (Stx3, ATP-binding cassette, sub-family A member 2 (Abca2, ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5, high mobility group nucleosomal binding domain 3 (Hmgn3 and pyruvate dehydrogenase beta (Pdhb. Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4.Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

  14. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

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    Yuan Li

    2017-02-01

    Full Text Available We aimed to investigate the effect of advanced glycation end products (AGEs on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3 II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  15. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.

    Science.gov (United States)

    Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

    2017-02-17

    We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2'-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  16. Inhibition of cathepsin K reduces cartilage degeneration in the anterior cruciate ligament transection rabbit and murine models of osteoarthritis.

    Science.gov (United States)

    Hayami, Tadashi; Zhuo, Ya; Wesolowski, Gregg A; Pickarski, Maureen; Duong, Le T

    2012-06-01

    To investigate the disease modifying effects of cathepsin K (CatK) inhibitor L-006235 compared to alendronate (ALN) in two preclinical models of osteoarthritis (OA). Skeletally mature rabbits underwent sham or anterior cruciate ligament transection (ACLT)-surgery and were treated with L-006235 (L-235, 10 mg/kg or 50 mg/kg, p.o., daily) or ALN (0.6 mg/kg, s.c., weekly) for 8-weeks. ACLT joint instability was also induced in CatK(-/-) versus wild type (wt) mice and treated for 16-weeks. Changes in cartilage degeneration, subchondral bone volume and osteophyte area were determined by histology and μ-CT. Collagen type I helical peptide (HP-I), a bone resorption marker and collagen type II C-telopeptide (CTX-II), a cartilage degradation marker were measured. L-235 (50 mg/kg) and ALN treatment resulted in significant chondroprotective effects, reducing CTX-II by 60% and the histological Mankin score for cartilage damage by 46% in the ACLT-rabbits. Both doses of L-235 were more potent than ALN in protecting against focal subchondral bone loss, and reducing HP-I by 70% compared to vehicle. L-235 (50 mg/kg) and ALN significantly reduced osteophyte formation in histomorphometric analysis by 55%. The Mankin score in ACLT-CatK(-/-) mice was ~2.5-fold lower than the ACLT-wt mice and was not different from sham-CatK(-/-). Osteophyte development was not different among the groups. Inhibition of CatK provides significant benefits in ACLT-model of OA, including: 1) protection of subchondral bone integrity, 2) protection against cartilage degradation and 3) reduced osteophytosis. Preclinical evidence supports the role of CatK as a potential therapeutic target for the treatment of OA. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Mutation analysis of the cathepsin C gene in Indian families with Papillon-Lefèvre syndrome

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    Srivastava Satish

    2003-07-01

    Full Text Available Abstract Background PLS is a rare autosomal recessive disorder characterized by early onset periodontopathia and palmar plantar keratosis. PLS is caused by mutations in the cathepsin C (CTSC gene. Dipeptidyl-peptidase I encoded by the CTSC gene removes dipeptides from the amino-terminus of protein substrates and mainly plays an immune and inflammatory role. Several mutations have been reported in this gene in patients from several ethnic groups. We report here mutation analysis of the CTSC gene in three Indian families with PLS. Methods Peripheral blood samples were obtained from individuals belonging to three Indian families with PLS for genomic DNA isolation. Exon-specific intronic primers were used to amplify DNA samples from individuals. PCR products were subsequently sequenced to detect mutations. PCR-SCCP and ASOH analyses were used to determine if mutations were present in normal control individuals. Results All patients from three families had a classic PLS phenotype, which included palmoplantar keratosis and early-onset severe periodontitis. Sequence analysis of the CTSC gene showed three novel nonsense mutations (viz., p.Q49X, p.Q69X and p.Y304X in homozygous state in affected individuals from these Indian families. Conclusions This study reported three novel nonsense mutations in three Indian families. These novel nonsense mutations are predicted to produce truncated dipeptidyl-peptidase I causing PLS phenotype in these families. A review of the literature along with three novel mutations reported here showed that the total number of mutations in the CTSC gene described to date is 41 with 17 mutations being located in exon 7.

  18. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

    International Nuclear Information System (INIS)

    Liow, K.Y.; Chow, S.C.

    2013-01-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration

  19. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    Science.gov (United States)

    Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

    2013-01-01

    Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

  20. The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease.

    Science.gov (United States)

    Gonzales Santana, Bibiana; Dalton, John P; Vasquez Camargo, Fabio; Parkinson, Michael; Ndao, Momar

    2013-01-01

    Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

  1. Plasma cathepsin S and cystatin C levels and risk of abdominal aortic aneurysm: a randomized population-based study.

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    Bing-Jie Lv

    Full Text Available BACKGROUND: Human abdominal aortic aneurysm (AAA lesions contain high levels of cathepsin S (CatS, but are deficient in its inhibitor, cystatin C. Whether plasma CatS and cystatin C levels are also altered in AAA patients remains unknown. METHODS AND RESULTS: Plasma samples were collected from 476 male AAA patients and 200 age-matched male controls to determine CatS and cystatin C levels by ELISA. Student's t test demonstrated higher plasma levels of total, active, and pro-CatS in AAA patients than in controls (P<0.001. ROC curve analysis confirmed higher plasma total, active, and pro-CatS levels in AAA patients than in controls (P<0.001. Logistic regression suggested that plasma total (odds ratio [OR] = 1.332, active (OR = 1.21, and pro-CatS (OR = 1.25 levels were independent AAA risk factors that associated positively with AAA (P<0.001. Plasma cystatin C levels associated significantly, but negatively, with AAA (OR = 0.356, P<0.001. Univariate correlation demonstrated that plasma total and active CatS levels correlated positively with body-mass index, diastolic blood pressure, and aortic diameter, but negatively with the lowest ankle-brachial index (ABI. Plasma cystatin C levels also correlated negatively with the lowest ABI. Multivariate linear regression showed that plasma total, active, and pro-CatS levels correlated positively with aortic diameter and negatively with the lowest ABI, whereas plasma cystatin C levels correlated negatively with aortic diameter and the lowest ABI, after adjusting for common AAA risk factors. CONCLUSIONS: Correlation of plasma CatS and cystatin C with aortic diameter and the lowest ABI suggest these serological parameters as biomarkers for human peripheral arterial diseases and AAA.

  2. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

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    Liow, K.Y.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

    2013-11-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

  3. Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

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    Ileana Corvo

    2018-04-01

    Full Text Available Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature. Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

  4. Differential expression levels of collagen 1A2, tissue inhibitor of metalloproteinase 4, and cathepsin B in intracranial aneurysms.

    Science.gov (United States)

    Babu, R Arun; Paul, Pradip; Purushottam, Meera; Srinivas, Dwarakanath; Somanna, Sampath; Jain, Sanjeev

    2016-01-01

    Intracranial aneurysms (IAs) express a variety of differentially expressed genes when compared to the normal artery. The aim of this study was to evaluate the expression level of a few genes in the aneurysm wall and to correlate them with various clinicoradiological factors. The mRNA level of collagen 1A2 (COL1A2), tissue inhibitor of metalloproteinase 4 (TIMP4), and cathepsin B (CTSB) genes were studied in 23 aneurysmal walls and 19 superficial temporal arteries harvested from 23 patients undergoing clipping of IAs, by real-time polymerase chain reaction method. The mean fold change of COL1A2 gene between the aneurysm sample and the superficial temporal artery (STA) sample was 2.46 ± 0.12, that of TIMP4 gene was 0.31 ± 0, and that of CTSB gene was 31.47 ± 39.01. There was a positive correlation of TIMP4 expression level with maximum diameter of aneurysm (P = 0.008) and fundus of aneurysm (P = 0.012). The mean fold change of CTSB of patients who had preoperative hydrocephalus in the computed tomogram (CT) scan of the head at admission was 56.16 and that of the patients who did not have hydrocephalus was 13.51 (P = 0.008). The mean fold change of CTSB of patients who developed fresh postoperative deficits or worsening of the preexisting deficits was 23.64 and that of the patients who did not develop was 42.22 (P = 0.039). COL1A2 gene and CTSB genes were overexpressed, and TIMP4 gene was underexpressed in the aneurysmal sac compared to STA and their expression levels were associated with a few clinicoradiological factors.

  5. 177Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model

    International Nuclear Information System (INIS)

    Ogbomo, Sunny M.; Shi, Wen; Wagh, Nilesh K.; Zhou, Zhengyuan; Brusnahan, Susan K.; Garrison, Jered C.

    2013-01-01

    Introduction: A major barrier to the advancement of therapeutic nanomedicines has been the non-target toxicity caused by the accumulation of the drug delivery systems in organs associated with the reticuloendothelial system, particularly the liver and spleen. Herein, we report the development of peptide based metabolically active linkers (MALs) that are enzymatically cleaved by cysteine cathepsin B and S, two proteases highly expressed in the liver and spleen. The overall goal of this approach is to utilize the MALs to lower the non-target retention and toxicity of radiolabeled drug delivery systems, thus resulting in higher diagnostic and radiotherapeutic efficacy. Methods: In this study three MALs (MAL0, MAL1 and MAL2) were investigated. MAL1 and MAL2 are composed of known substrates of cathepsin B and S, respectively, while MAL0 is a non-cleavable control. Both MAL1 and MAL2 were shown to undergo enzymatic cleavage with the appropriate cathepsin protease. Subsequent to conjugation to the HPMA copolymer and radiolabeling with 177 Lu, the peptide–polymer conjugates were renamed 177 Lu-metabolically active copolymers ( 177 Lu-MACs) with the corresponding designations: 177 Lu-MAC0, 177 Lu-MAC1 and 177 Lu-MAC2. Results: In vivo evaluation of the 177 Lu-MACs was performed in an HPAC human pancreatic cancer xenograft mouse model. 177 Lu-MAC1 and 177 Lu-MAC2 demonstrated 3.1 and 2.1 fold lower liver retention, respectively, compared to control ( 177 Lu-MAC0) at 72 h post-injection. With regard to spleen retention, 177 Lu-MAC1 and 177 Lu-MAC2 each exhibited a nearly fourfold lower retention, relative to control, at the 72 h time point. However, the tumor accumulation of the 177 Lu-MAC0 was two to three times greater than 177 Lu-MAC1 and 177 Lu-MAC2 at the same time point. The MAL approach demonstrated the capability of substantially reducing the non-target retention of the 177 Lu-labeled HPMA copolymers. Conclusions: While further studies are needed to optimize the

  6. Synaptic changes in the thalamocortical system of cathepsin D-deficient mice: a model of human congenital neuronal ceroid-lipofuscinosis.

    Science.gov (United States)

    Partanen, Sanna; Haapanen, Aleksi; Kielar, Catherine; Pontikis, Charles; Alexander, Noreen; Inkinen, Teija; Saftig, Paul; Gillingwater, Thomas H; Cooper, Jonathan D; Tyynelä, Jaana

    2008-01-01

    Cathepsin D (CTSD; EC 3.4.23.5) is a lysosomal aspartic protease, the deficiency of which causes early-onset and particularly aggressive forms of neuronal ceroid-lipofuscinosis in infants, sheep, and mice. Cathepsin D deficiencies are characterized by severe neurodegeneration, but the molecular mechanisms behind the neuronal death remain poorly understood. In this study, we have systematically mapped the distribution of neuropathologic changes in CTSD-deficient mouse brains by stereologic, immunologic, and electron microscopic methods. We report highly accentuated neuropathologic changes within the ventral posterior nucleus (ventral posteromedial [VPM]/ventral posterolateral [VPL]) of thalamus and in neuronal laminae IV and VI of the somatosensory cortex (S1BF), which receive and send information to the thalamic VPM/VPL. These changes included pronounced astrocytosis and microglial activation that begin in the VPM/VPL thalamic nucleus of CTSD-deficient mice and are associated with reduced neuronal number and redistribution of presynaptic markers. In addition, loss of synapses, axonal pathology, and aggregation of synaptophysin and synaptobrevin were observed in the VPM/VPL. These synaptic alterations are accompanied by changes in the amount of synaptophysin/synaptobrevin heterodimer, which regulates formation of the SNARE complex at the synapse. Taken together, these data reveal the somatosensory thalamocortical circuitry as a particular focus of pathologic changes and provide the first evidence for synaptic alterations at the molecular and ultrastructural levels in CTSD deficiency.

  7. Impact of cathepsin B-sensitive triggers and hydrophilic linkers on in vitro efficacy of novel site-specific antibody-drug conjugates.

    Science.gov (United States)

    Bryden, Francesca; Martin, Camille; Letast, Stéphanie; Lles, Eva; Viéitez-Villemin, Inmaculada; Rousseau, Anaïs; Colas, Cyril; Brachet-Botineau, Marie; Allard-Vannier, Emilie; Larbouret, Christel; Viaud-Massuard, Marie-Claude; Joubert, Nicolas

    2018-03-14

    Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG 12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

  8. A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins

    Science.gov (United States)

    Diaz, Isabel

    2012-01-01

    Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described PMID:22791822

  9. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone

    2004-01-01

    been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose......-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors z...

  10. The Feasibility of Enzyme Targeted Activation for Amino Acid/Dipeptide Monoester Prodrugs of Floxuridine; Cathepsin D as a Potential Targeted Enzyme

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    Gordon L. Amidon

    2012-03-01

    Full Text Available The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5¢-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0–105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5¢-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine and 5¢-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enyzmes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  11. The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

    Science.gov (United States)

    Tsume, Yasuhiro; Amidon, Gordon L

    2012-03-26

    The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  12. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

    Directory of Open Access Journals (Sweden)

    Daniel Menezes-Souza

    2015-01-01

    Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

  13. Multiple Perspectives / Multiple Readings

    Directory of Open Access Journals (Sweden)

    Simon Biggs

    2005-01-01

    Full Text Available People experience things from their own physical point of view. What they see is usually a function of where they are and what physical attitude they adopt relative to the subject. With augmented vision (periscopes, mirrors, remote cameras, etc we are able to see things from places where we are not present. With time-shifting technologies, such as the video recorder, we can also see things from the past; a time and a place we may never have visited.In recent artistic work I have been exploring the implications of digital technology, interactivity and internet connectivity that allow people to not so much space/time-shift their visual experience of things but rather see what happens when everybody is simultaneously able to see what everybody else can see. This is extrapolated through the remote networking of sites that are actual installation spaces; where the physical movements of viewers in the space generate multiple perspectives, linked to other similar sites at remote locations or to other viewers entering the shared data-space through a web based version of the work.This text explores the processes involved in such a practice and reflects on related questions regarding the non-singularity of being and the sense of self as linked to time and place.

  14. Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

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    Ileana Corvo

    Full Text Available A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2.Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS, we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL. Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3.These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage

  15. The C-terminal subunit of artificially truncated human cathepsin B mediates its nuclear targeting and contributes to cell viability

    Directory of Open Access Journals (Sweden)

    Dallner Claudia

    2005-04-01

    Full Text Available Abstract Background Splicing variants of human cathepsinB primary transcripts (CB(-2,3 result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also

  16. Rheumatoid Factor Positivity Is Associated with Increased Joint Destruction and Upregulation of Matrix Metalloproteinase 9 and Cathepsin K Gene Expression in the Peripheral Blood in Rheumatoid Arthritic Patients Treated with Methotrexate

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    Elena V. Tchetina

    2013-01-01

    Full Text Available We evaluated changes in gene expression of mTOR, p21, caspase-3, ULK1, TNFα, matrix metalloproteinase (MMP-9, and cathepsin K in the whole blood of rheumatoid arthritic (RA patients treated with methotrexate (MTX in relation to their rheumatoid factor status, clinical, immunological, and radiological parameters, and therapeutic response after a 24-month follow-up. The study group consisted of 35 control subjects and 33 RA patients without previous history of MTX treatment. Gene expression was measured using real-time RT-PCR. Decreased disease activity in patients at the end of the study was associated with significant downregulation of TNFα expression. Downregulation of mTOR was observed in seronegative patients, while no significant changes in the expression of p21, ULK1, or caspase-3 were noted in any RA patients at the end of the study. The increase in erosion numbers observed in the seropositive patients at the end of the follow-up was accompanied by upregulation of MMP-9 and cathepsin K, while seronegative patients demonstrated an absence of significant changes in MMP-9 and cathepsin K expression and no increase in the erosion score. Our results suggest that increased expression of MMP-9 and cathepsin K genes in the peripheral blood might indicate higher bone tissue destruction activity in RA patients treated with methotrexate. The clinical study registration number is 0120.0810610.

  17. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

    Science.gov (United States)

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H.; Gutiérrez, Andrés H.; Rueda, Luis D.; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H.; Sheen, Patricia

    2011-01-01

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection. PMID:22119017

  18. Multiple sclerosis

    Science.gov (United States)

    ... indwelling catheter Osteoporosis or thinning of the bones Pressure sores Side effects of medicines used to treat the ... Daily bowel care program Multiple sclerosis - discharge Preventing pressure ulcers Swallowing problems Images Multiple sclerosis MRI of the ...

  19. MULTIPLE OBJECTS

    Directory of Open Access Journals (Sweden)

    A. A. Bosov

    2015-04-01

    Full Text Available Purpose. The development of complicated techniques of production and management processes, information systems, computer science, applied objects of systems theory and others requires improvement of mathematical methods, new approaches for researches of application systems. And the variety and diversity of subject systems makes necessary the development of a model that generalizes the classical sets and their development – sets of sets. Multiple objects unlike sets are constructed by multiple structures and represented by the structure and content. The aim of the work is the analysis of multiple structures, generating multiple objects, the further development of operations on these objects in application systems. Methodology. To achieve the objectives of the researches, the structure of multiple objects represents as constructive trio, consisting of media, signatures and axiomatic. Multiple object is determined by the structure and content, as well as represented by hybrid superposition, composed of sets, multi-sets, ordered sets (lists and heterogeneous sets (sequences, corteges. Findings. In this paper we study the properties and characteristics of the components of hybrid multiple objects of complex systems, proposed assessments of their complexity, shown the rules of internal and external operations on objects of implementation. We introduce the relation of arbitrary order over multiple objects, we define the description of functions and display on objects of multiple structures. Originality.In this paper we consider the development of multiple structures, generating multiple objects.Practical value. The transition from the abstract to the subject of multiple structures requires the transformation of the system and multiple objects. Transformation involves three successive stages: specification (binding to the domain, interpretation (multiple sites and particularization (goals. The proposed describe systems approach based on hybrid sets

  20. Mycobacterium tuberculosis Modulates miR-106b-5p to Control Cathepsin S Expression Resulting in Higher Pathogen Survival and Poor T-Cell Activation

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    David Pires

    2017-12-01

    Full Text Available The success of tuberculosis (TB bacillus, Mycobacterium tuberculosis (Mtb, relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3′-untranslated region of cathepsin S (CtsS mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.

  1. Association between polymorphism in the human cathepsin L (CTSL 1) promoter with hypertension in the uygur, kazak and han population in china

    International Nuclear Information System (INIS)

    Chen, S.; Wang, Z.; Zhou, C.; Wang, D.W.

    2015-01-01

    To systemically investigate the association between the polymorphism (rs3118869) in cathepsin Lenzyme gene with hypertension in three ethnic groups (Han, Kazak and Uygur) in China. Study Design: Case-control study. Place and Duration of Study: Department of Cardiology, The First Affiliated Hospital, Shihezi Medical College, Shihezi University and Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2013 to May 2014. Methodology: This case-control study included 1224 patients (422 Uygur, 425 Kazak and 377 Han individuals) with hypertension and 967 healthy unrelated individuals (339 Uygur, 337 Kazak and 291 Han individuals) as controls. The participants came from three ethnic groups (Han, Kazak and Uygur) which were recruited from Xinjiang Province of China. The polymorphism (rs3118869) of the human cathepsin Lgene was genotyped using the TaqMan 5' nuclease assay. Binary logistic regression was built to determine the association of polymorphism with hypertension. Results: The genotype distribution of polymorphism was not significantly different in three ethnic groups. The rs3118869 polymorphism was significantly associated with Essential Hypertension (EH) in co-dominant model (A/C vs. C/C) in total people (OR = 0.697, 95% CI = 0.520 -0.932, p = 0.015), the same result was obtained in recessive model (C/C + A/C vs. A/A) in total people (OR = 0.689, 95% CI = 0.522 -0.910, p = 0.009). Similar finding of rs3118869 in recessive model (C/C + A/C vs. A/A) was also observed after adjusting the variable to the covariates age (OR = 0.629, 95% CI = 0.464 0853, p = 0.003). Conclusion: The study results indicate the A-allele of rs3118869 is a protective factor in hypertension. (author)

  2. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

    Science.gov (United States)

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

  3. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    Science.gov (United States)

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  4. Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

    Science.gov (United States)

    Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

    2015-07-01

    Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.

  5. Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus: A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection.

    Science.gov (United States)

    Wang, Guobao; Zhang, Jianjia; Shen, Yunwang; Zheng, Qin; Feng, Min; Xiang, Xingwei; Wu, Xiaofeng

    2015-06-01

    Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B. mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B. mori. Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV): Natural occurrence and efficacy as a biological insecticide on young banana plants in greenhouse and open-field conditions on the Canary Islands.

    Science.gov (United States)

    Fuentes, Ernesto Gabriel; Hernández-Suárez, Estrella; Simón, Oihane; Williams, Trevor; Caballero, Primitivo

    2017-01-01

    Chrysodeixis chalcites, an important pest of banana crops on the Canary Islands, is usually controlled by chemical insecticides. The present study aimed to evaluate the efficacy of the most prevalent isolate of the Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV, Baculoviridae) as a biological insecticide. Overall the prevalence of ChchNPV infection in C. chalcites populations was 2.3% (103 infected larvae out of 4,438 sampled), but varied from 0-4.8% on Tenerife and was usually low (0-2%) on the other islands. On Tenerife, infected larvae were present at 11 out of 17 plantations sampled. The prevalence of infection in larvae on bananas grown under greenhouse structures was significantly higher (3%) than in open-field sites (1.4%). The ChchNPV-TF1 isolate was the most abundant and widespread of four genetic variants of the virus. Application of 1.0x109 viral occlusion bodies (OBs)/l of ChchNPV-TF1 significantly reduced C. chalcites foliar damage in young banana plants as did commonly used pesticides, both in greenhouse and open-field sites. The insecticidal efficacy of ChchNPV-TF1 was similar to that of indoxacarb and a Bacillus thuringiensis (Bt)-based insecticide in one year of trials and similar to Bt in the following year of trails in greenhouse and field crops. However, larvae collected at different time intervals following virus treatments and reared in the laboratory experienced 2-7 fold more mortality than insects from conventional insecticide treatments. This suggests that the acquisition of lethal dose occurred over an extended period (up to 7 days) compared to a brief peak in larvae on plants treated with conventional insecticides. These results should prove useful for the registration of a ChchNPV-based insecticide for integrated management of this pest in banana crops on the Canary Islands.

  7. Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV: Natural occurrence and efficacy as a biological insecticide on young banana plants in greenhouse and open-field conditions on the Canary Islands.

    Directory of Open Access Journals (Sweden)

    Ernesto Gabriel Fuentes

    Full Text Available Chrysodeixis chalcites, an important pest of banana crops on the Canary Islands, is usually controlled by chemical insecticides. The present study aimed to evaluate the efficacy of the most prevalent isolate of the Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV, Baculoviridae as a biological insecticide. Overall the prevalence of ChchNPV infection in C. chalcites populations was 2.3% (103 infected larvae out of 4,438 sampled, but varied from 0-4.8% on Tenerife and was usually low (0-2% on the other islands. On Tenerife, infected larvae were present at 11 out of 17 plantations sampled. The prevalence of infection in larvae on bananas grown under greenhouse structures was significantly higher (3% than in open-field sites (1.4%. The ChchNPV-TF1 isolate was the most abundant and widespread of four genetic variants of the virus. Application of 1.0x109 viral occlusion bodies (OBs/l of ChchNPV-TF1 significantly reduced C. chalcites foliar damage in young banana plants as did commonly used pesticides, both in greenhouse and open-field sites. The insecticidal efficacy of ChchNPV-TF1 was similar to that of indoxacarb and a Bacillus thuringiensis (Bt-based insecticide in one year of trials and similar to Bt in the following year of trails in greenhouse and field crops. However, larvae collected at different time intervals following virus treatments and reared in the laboratory experienced 2-7 fold more mortality than insects from conventional insecticide treatments. This suggests that the acquisition of lethal dose occurred over an extended period (up to 7 days compared to a brief peak in larvae on plants treated with conventional insecticides. These results should prove useful for the registration of a ChchNPV-based insecticide for integrated management of this pest in banana crops on the Canary Islands.

  8. Multiple sclerosis

    International Nuclear Information System (INIS)

    Grunwald, I.Q.; Kuehn, A.L.; Backens, M.; Papanagiotou, P.; Shariat, K.; Kostopoulos, P.

    2008-01-01

    Multiple sclerosis is the most common chronic inflammatory disease of myelin with interspersed lesions in the white matter of the central nervous system. Magnetic resonance imaging (MRI) plays a key role in the diagnosis and monitoring of white matter diseases. This article focuses on key findings in multiple sclerosis as detected by MRI. (orig.) [de

  9. Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

    Science.gov (United States)

    Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

    Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

  10. Multiple homicides.

    Science.gov (United States)

    Copeland, A R

    1989-09-01

    A study of multiple homicides or multiple deaths involving a solitary incident of violence by another individual was performed on the case files of the Office of the Medical Examiner of Metropolitan Dade County in Miami, Florida, during 1983-1987. A total of 107 multiple homicides were studied: 88 double, 17 triple, one quadruple, and one quintuple. The 236 victims were analyzed regarding age, race, sex, cause of death, toxicologic data, perpetrator, locale of the incident, and reason for the incident. This article compares this type of slaying with other types of homicide including those perpetrated by serial killers. Suggestions for future research in this field are offered.

  11. Multiple Sclerosis

    Science.gov (United States)

    Multiple sclerosis (MS) is a nervous system disease that affects your brain and spinal cord. It damages the myelin sheath, the material that surrounds and protects your nerve cells. This damage slows down ...

  12. Multiple myeloma.

    LENUS (Irish Health Repository)

    Collins, Conor D

    2012-02-01

    Advances in the imaging and treatment of multiple myeloma have occurred over the past decade. This article summarises the current status and highlights how an understanding of both is necessary for optimum management.

  13. Multiple mononeuropathy

    Science.gov (United States)

    ... with multiple mononeuropathy are prone to new nerve injuries at pressure points such as the knees and elbows. They should avoid putting pressure on these areas, for example, by not leaning on the elbows, crossing the knees, ...

  14. Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin.

    Science.gov (United States)

    Mancilla-Olea, Maria Inocente; Ortega-López, Jaime; Figueroa-Angulo, Elisa E; Avila-González, Leticia; Cárdenas-Guerra, Rosa Elena; Miranda-Ozuna, Jesús F T; González-Robles, Arturo; Hernández-García, Mar Saraí; Sánchez-Ayala, Lizbeth; Arroyo, Rossana

    2018-04-01

    Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis.

    Science.gov (United States)

    Meshgi, Behnam; Jalousian, Fatemeh; Fathi, Saeid; Jahani, Zahra

    2018-06-01

    Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative

  16. [Multiple meningiomas].

    Science.gov (United States)

    Terrier, L-M; François, P

    2016-06-01

    Multiple meningiomas (MMs) or meningiomatosis are defined by the presence of at least 2 lesions that appear simultaneously or not, at different intracranial locations, without the association of neurofibromatosis. They present 1-9 % of meningiomas with a female predominance. The occurrence of multiple meningiomas is not clear. There are 2 main hypotheses for their development, one that supports the independent evolution of these tumors and the other, completely opposite, that suggests the propagation of tumor cells of a unique clone transformation, through cerebrospinal fluid. NF2 gene mutation is an important intrinsic risk factor in the etiology of multiple meningiomas and some exogenous risk factors have been suspected but only ionizing radiation exposure has been proven. These tumors can grow anywhere in the skull but they are more frequently observed in supratentorial locations. Their histologic types are similar to unique meningiomas of psammomatous, fibroblastic, meningothelial or transitional type and in most cases are benign tumors. The prognosis of these tumors is eventually good and does not differ from the unique tumors except for the cases of radiation-induced multiple meningiomas, in the context of NF2 or when diagnosed in children where the outcome is less favorable. Each meningioma lesion should be dealt with individually and their multiple character should not justify their resection at all costs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Multiple sclerosis

    DEFF Research Database (Denmark)

    Stenager, Egon; Stenager, E N; Knudsen, Lone

    1994-01-01

    In a cross-sectional study of 117 randomly selected patients (52 men, 65 women) with definite multiple sclerosis, it was found that 76 percent were married or cohabitant, 8 percent divorced. Social contacts remained unchanged for 70 percent, but outgoing social contacts were reduced for 45 percent......, need for structural changes in home and need for pension became greater with increasing physical handicap. No significant differences between gender were found. It is concluded that patients and relatives are under increased social strain, when multiple sclerosis progresses to a moderate handicap...

  18. Multiple myeloma

    International Nuclear Information System (INIS)

    Sohn, Jeong Ick; Ha, Choon Ho; Choi, Karp Shik

    1994-01-01

    Multiple myeloma is a malignant plasma cell tumor that is thought to originate proliferation of a single clone of abnormal plasma cell resulting production of a whole monoclonal paraprotein. The authors experienced a case of multiple myeloma with severe mandibular osteolytic lesions in 46-year-old female. As a result of careful analysis of clinical, radiological, histopathological features, and laboratory findings, we diagnosed it as multiple myeloma, and the following results were obtained. 1. Main clinical symptoms were intermittent dull pain on the mandibular body area, abnormal sensation of lip and pain due to the fracture on the right clavicle. 2. Laboratory findings revealed M-spike, reversed serum albumin-globulin ratio, markedly elevated ESR and hypercalcemia. 3. Radiographically, multiple osteolytic punched-out radiolucencies were evident on the skull, zygoma, jaw bones, ribs, clavicle and upper extremities. Enlarged liver and increased uptakes on the lesional sites in RN scan were also observed. 4. Histopathologically, markedly hypercellular marrow with sheets of plasmoblasts and megakaryocytes were also observed.

  19. Multiple sclerosis

    DEFF Research Database (Denmark)

    Stenager, E; Jensen, K

    1988-01-01

    Forty-two (12%) of a total of 366 patients with multiple sclerosis (MS) had psychiatric admissions. Of these, 34 (81%) had their first psychiatric admission in conjunction with or after the onset of MS. Classification by psychiatric diagnosis showed that there was a significant positive correlation...

  20. Multiple sclerosis

    DEFF Research Database (Denmark)

    Stenager, E; Knudsen, L; Jensen, K

    1991-01-01

    In a cross-sectional investigation of 116 patients with multiple sclerosis, the social and sparetime activities of the patient were assessed by both patient and his/her family. The assessments were correlated to physical disability which showed that particularly those who were moderately disabled...

  1. Multiple sclerosis

    DEFF Research Database (Denmark)

    Stenager, E; Jensen, K

    1990-01-01

    An investigation on the correlation between ability to read TV subtitles and the duration of visual evoked potential (VEP) latency in 14 patients with definite multiple sclerosis (MS), indicated that VEP latency in patients unable to read the TV subtitles was significantly delayed in comparison...

  2. Multiple sclerosis

    DEFF Research Database (Denmark)

    Stenager, E; Knudsen, L; Jensen, K

    1994-01-01

    In a cross-sectional study of 94 patients (42 males, 52 females) with definite multiple sclerosis (MS) in the age range 25-55 years, the correlation of neuropsychological tests with the ability to read TV-subtitles and with the use of sedatives is examined. A logistic regression analysis reveals...

  3. Multiple Sclerosis.

    Science.gov (United States)

    Plummer, Nancy; Michael, Nancy, Ed.

    This module on multiple sclerosis is intended for use in inservice or continuing education programs for persons who administer medications in long-term care facilities. Instructor information, including teaching suggestions, and a listing of recommended audiovisual materials and their sources appear first. The module goal and objectives are then…

  4. Parenting Multiples

    Science.gov (United States)

    ... when your babies do. Though it can be hard to let go of the thousand other things you need to do, remember that your well-being is key to your ability to take care of your babies. What Problems Can Happen? It may be hard to tell multiple babies apart when they first ...

  5. Repeated oral administration of a cathepsin K inhibitor significantly suppresses bone resorption in exercising horses with evidence of increased bone formation and maintained bone turnover.

    Science.gov (United States)

    Hussein, H; Dulin, J; Smanik, L; Drost, W T; Russell, D; Wellman, M; Bertone, A

    2017-08-01

    Our investigations evaluated the effect of VEL-0230, a highly specific irreversible inhibitor of cathepsin K (CatK). The objectives of our study were to determine whether repeated dosing of a CatK inhibitor (CatKI) produced a desired inhibition of the bone resorption biomarker (CTX-1), and document the effect of repeated dosing on bone homeostasis, structure, and dynamics of bone resorption and formation in horses. Twelve young exercising horses were randomized in a prospective, controlled clinical trial and received 4 weekly doses of a CatKI or vehicle. Baseline and poststudy nuclear scintigraphy, blood sampling and analysis of plasma bone biomarkers (CTX-1 and osteocalcin), poststudy bone fluorescent labeling, and bone biopsy were performed. Bone specimens were further processed for microcomputed tomography and bone histomorphometry. Each dose of this CatKI transiently inhibited plasma CTX-1 (reflecting inhibition of bone collagen resorption) and increased bone plasma osteocalcin concentrations, with no detectable adverse effect on normal bone turnover in the face of exercise. Bone morphology, density, and formation rate were not different between control and treated group. Further investigation of CatK inhibition in abnormal bone turnover is required in animals with bone diseases. © 2016 John Wiley & Sons Ltd.

  6. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Directory of Open Access Journals (Sweden)

    Bruno Gottstein

    2014-06-01

    Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

  7. Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia.

    Science.gov (United States)

    Paradiso, A; Mangia, A; Correale, M; Abbate, I; Ferri, G; Piffanelli, A; Catozzi, L; Amadori, D; Riccobon, A; De Lena, M

    1992-01-01

    Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.

  8. Whole-exome sequencing reveals a recurrent mutation in the cathepsin C gene that causes Papillon–Lefevre syndrome in a Saudi family

    Directory of Open Access Journals (Sweden)

    Yaser Mohammad Alkhiary

    2016-09-01

    Full Text Available Papillon–Lefevre syndrome (PALS is a rare, autosomal recessive disorder characterized by periodontitis and hyperkeratosis over the palms and soles. Mutations in the cathepsin C gene (CTSC have been recognized as the cause of PALS since the late 1990s. More than 75 mutations in CTSC have been identified, and phenotypic variability between different mutations has been described. Next generation sequencing is widely used for efficient molecular diagnostics in various clinical practices. Here we investigated a large consanguineous Saudi family with four affected and four unaffected individuals. All of the affected individuals suffered from hyperkeratosis over the palms and soles and had anomalies of both primary and secondary dentition. For molecular diagnostics, we combined whole-exome sequencing and genome-wide homozygosity mapping procedures, and identified a recurrent homozygous missense mutation (c.899G>A; p.Gly300Asp in exon 7 of CTSC. Validation of all eight family members by Sanger sequencing confirmed co-segregation of the pathogenic variant (c.899G>A with the disease phenotype. This is the first report of whole-exome sequencing performed for molecular diagnosis of PALS in Saudi Arabia. Our findings provide further insights into the genotype–phenotype correlation of CTSC pathogenicity in PALS.

  9. Tamm-Horsfall Glycoprotein Enhances PMN Phagocytosis by Binding to Cell Surface-Expressed Lactoferrin and Cathepsin G That Activates MAP Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Chia-Li Yu

    2011-03-01

    Full Text Available The molecular basis of polymorphonuclear neutrophil (PMN phagocytosis-enhancing activity (PEA by human purified urinary Tamm-Horsfall glyco- protein (THP has not been elucidated. In this study, we found human THP bound to lactoferrin (LF and cathepsin G (CG expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-kB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase, protein specificity (V8 protease and proteinase K or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase. We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

  10. Multiple sclerosis

    International Nuclear Information System (INIS)

    Sadashima, Hiromichi; Kusaka, Hirofumi; Imai, Terukuni; Takahashi, Ryosuke; Matsumoto, Sadayuki; Yamamoto, Toru; Yamasaki, Masahiro; Maya, Kiyomi

    1986-01-01

    Eleven patients with a definite diagnosis of multiple sclerosis were examined in terms of correlations between the clinical features and the results of cranial computed tomography (CT), and magnetic resonance imaging (MRI). Results: In 5 of the 11 patients, both CT and MRI demonstrated lesions consistent with a finding of multiple sclerosis. In 3 patients, only MRI demonstrated lesions. In the remaining 3 patients, neither CT nor MRI revealed any lesion in the brain. All 5 patients who showed abnormal findings on both CT and MRI had clinical signs either of cerebral or brainstem - cerebellar lesions. On the other hand, two of the 3 patients with normal CT and MRI findings had optic-nerve and spinal-cord signs. Therefore, our results suggested relatively good correlations between the clinical features, CT, and MRI. MRI revealed cerebral lesions in two of the four patients with clinical signs of only optic-nerve and spinal-cord lesions. MRI demonstrated sclerotic lesions in 3 of the 6 patients whose plaques were not detected by CT. In conclusion, MRI proved to be more helpful in the demonstration of lesions attributable to chronic multiple sclerosis. (author)

  11. Multiple inflation

    International Nuclear Information System (INIS)

    Murphy, P.J.

    1987-01-01

    The Theory of Inflation, namely, that at some point the entropy content of the universe was greatly increased, has much promise. It may solve the puzzles of homogeneity and the creation of structure. However, no particle physics model has yet been found that can successfully drive inflation. The difficulty in satisfying the constraint that the isotropy of the microwave background places on the effective potential of prospective models is immense. In this work we have codified the requirements of such models in a most general form. We have carefully calculated the amounts of inflation the various problems of the Standard Model need for their solution. We have derived a completely model independent upper bond on the inflationary Hubble parameter. We have developed a general notation with which to probe the possibilities of Multiple Inflation. We have shown that only in very unlikely circumstances will any evidence of an earlier inflation, survive the de Sitter period of its successor. In particular, it is demonstrated that it is most unlikely that two bouts of inflation will yield high amplitudes of density perturbations on small scales and low amplitudes on large. We conclude that, while multiple inflation will be of great theoretical interest, it is unlikely to have any observational impact

  12. Towards Delineating Functions within the Fasciola Secreted Cathepsin L Protease Family by Integrating In Vivo Based Sub-Proteomics and Phylogenetics

    Science.gov (United States)

    Morphew, Russell M.; Wright, Hazel A.; LaCourse, E. James; Porter, Joanne; Barrett, John; Woods, Debra J.; Brophy, Peter M.

    2011-01-01

    Background Fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family. Methodology/Principal Findings The sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. Conclusions/Significance We have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase. PMID:21245911

  13. Inhibition of Cathepsins B Induces Neuroprotection Against Secondary Degeneration in Ipsilateral Substantia Nigra After Focal Cortical Infarction in Adult Male Rats

    Directory of Open Access Journals (Sweden)

    Xialin Zuo

    2018-05-01

    Full Text Available Stroke is the leading cause of adult disability in the world. In general, recovery from stroke is incomplete. Accumulating evidences have shown that focal cerebral infarction leads to dynamic trans-neuronal degeneration in non-ischemic remote brain regions, with the disruption of connections to synapsed neurons sustaining ischemic insults. Previously, we had reported that the ipsilateral striatum, thalamus degenerated in succession after permanent distal branch of middle cerebral artery occlusion (dMCAO in Sprague-Dawley (SD rats and cathepsin (Cath B was activated before these relay degeneration. Here, we investigate the role of CathB in the secondary degeneration of ipsilateral substantia nigra (SN after focal cortical infarction. We further examined whether the inhibition of CathB with L-3-trans-(Propyl-carbamoyloxirane-2-carbonyl-L-isoleucyl-L-proline methyl ester (CA-074Me would attenuate secondary degeneration through enhancing the cortico-striatum-nigral connections and contribute to the neuroprotective effects. Our results demonstrated that secondary degeneration in the ipsilateral SN occurred and CathB was upregulated in the ipsilateral SN after focal cortical infarction. The inhibition of CathB with CA-074Me reduced the neuronal loss and gliosis in the ipsilateral SN. Using biotinylated dextran amine (BDA or pseudorabies virus (PRV 152 as anterograde or retrograde tracer to trace striatum-nigral and cortico-nigral projections pathway, CA-074Me can effectively enhance the cortico-striatum-nigral connections and exert neuroprotection against secondary degeneration in the ipsilateral SN after cortical ischemia. Our study suggests that the lysosomal protease CathB mediates the secondary damage in the ipsilateral SN after dMCAO, thus it can be a promising neuroprotective target for the rehabilitation of stroke patients.

  14. Suppression of Human T Cell Proliferation Mediated by the Cathepsin B Inhibitor, z-FA-FMK Is Due to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Tanuja Rajah

    Full Text Available The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-fluoromethyl ketone (z-FA-FMK readily inhibits anti-CD3-induced human T cell proliferation, whereas the analogue benzyloxycarbonyl-phenylalanine-alanine-diazomethyl ketone (z-FA-DMK had no effect. In contrast, benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK was toxic. The inhibition of T cell proliferation mediated by z-FA-FMK requires not only the FMK moiety, but also the benzyloxycarbonyl group at the N-terminal, suggesting some degree of specificity in z-FA-FMK-induced inhibition of primary T cell proliferation. We showed that z-FA-FMK treatment leads to a decrease in intracellular glutathione (GSH with a concomitant increase in reactive oxygen species (ROS levels in activated T cells. The inhibition of anti-CD3-induced T cell proliferation mediated by z-FA-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. The inhibition of anti-CD3-induced up-regulation of CD25 and CD69 expression mediated by z-FA-FMK was also attenuated in the presence of exogenous GSH. Similar to cell proliferation, GSH, NAC and L-cysteine but not D-cysteine, completely restored the processing of caspase-8 and caspase-3 to their respective subunits in z-FA-FMK-treated activated T cells. Our collective results demonstrated that the inhibition of T cell activation and proliferation mediated by z-FA-FMK is due to oxidative stress via the depletion of GSH.

  15. Towards delineating functions within the fasciola secreted cathepsin l protease family by integrating in vivo based sub-proteomics and phylogenetics.

    Directory of Open Access Journals (Sweden)

    Russell M Morphew

    2011-01-01

    Full Text Available fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES products are also the most promising vaccine candidates, the cathepsin L (Cat L protease family.the sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples.we have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.

  16. Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

    Science.gov (United States)

    Wolf, Marlene; Clark-Lewis, Ian; Buri, Caroline; Langen, Hanno; Lis, Maddalena; Mazzucchelli, Luca

    2003-01-01

    Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes. PMID:12651610

  17. Mice, double deficient in lysosomal serine carboxypeptidases Scpep1 and Cathepsin A develop the hyperproliferative vesicular corneal dystrophy and hypertrophic skin thickenings.

    Directory of Open Access Journals (Sweden)

    Xuefang Pan

    Full Text Available Vasoactive and mitogenic peptide, endothelin-1 (ET-1 plays an important role in physiology of the ocular tissues by regulating the growth of corneal epithelial cells and maintaining the hemodynamics of intraocular fluids. We have previously established that ET-1 can be degraded in vivo by two lysosomal/secreted serine carboxypeptidases, Cathepsin A (CathA and Serine Carboxypeptidase 1 (Scpep1 and that gene-targeted CathAS190A /Scpep1-/- mice, deficient in CathA and Scpep1 have a prolonged half-life of circulating ET-1 associated with systemic hypertension. In the current work we report that starting from 6 months of age, ~43% of CathAS190A /Scpep1-/- mice developed corneal clouding that eventually caused vision impairment. Histological evaluation of these mice demonstrated a selective fibrotic thickening and vacuolization of the corneas, resembling human hyperproliferative vesicular corneal stromal dystrophy and coexisting with a peculiar thickening of the skin epidermis. Moreover, we found that cultured corneal epithelial cells, skin fibroblasts and vascular smooth muscle cells derived from CathA/Scpep1-deficient mice, demonstrated a significantly higher proliferative response to treatment with exogenous ET-1, as compared with cells from wild type mice. We also detected increased activation level of ERK1/2 and AKT kinases involved in cell proliferation in the ET-1-treated cultured cells from CathA/Scpep1 deficient mice. Together, results from our experimental model suggest that; in normal tissues the tandem of serine carboxypeptidases, Scpep1 and CathA likely constitutes an important part of the physiological mechanism responsible for the balanced elimination of heightened levels of ET-1 that otherwise would accumulate in tissues and consequently contribute to development of the hyper-proliferative corneal dystrophy and abnormal skin thickening.

  18. Protective protein/cathepsin A down-regulates osteoclastogenesis by associating with and degrading NF-kappaB p50/p65.

    Science.gov (United States)

    Masuhara, Masaaki; Sato, Takuya; Hada, Naoto; Hakeda, Yoshiyuki

    2009-01-01

    Disruption of the cooperative function balance between osteoblasts and osteoclasts causes various bone disorders, some of which are attributed to abnormal osteoclast recruitment. Osteoclast differentiation is dependent on the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) as well as the macrophage colony-stimulating factor. The osteoclast formation induced by cytokines requires activation of NF-kappaB, AP-1 and nuclear factor of activated T cells c1. However, osteoclasts are not the only cell types that express these transcription factors, suggesting that some unknown molecules specific for osteoclasts may associate with the transcription factors. Here, we explored the possibility of molecules binding directly to NF-kappaB and cloned protective protein/cathepsin A (PPCA) by yeast two-hybrid screening using a cDNA library of osteoclast precursors. Forced expression of PPCA with p50/p65 in HEK293 cells decreased both the level of p50/p65 proteins and the transcriptional activity. Abundant PPCA was detected in the lysosomes of the transfected HEK293 cells, but a small amount of this enzyme was also present in the cytosolic fraction. In addition, over-expression of PPCA caused the disappearance of p50/p65 in both the lysosomal and cytosolic fractions. PPCA was expressed throughout osteoclastogenesis, and the expression was slightly up-regulated by RANKL signaling. Knockdown of PPCA in osteoclast precursors with PPCA siRNA stimulated binding of nuclear proteins to oligonucleotides containing an NF-kappaB binding motif and increased osteoclastogenesis. Our present results indicate a novel role for PPCA in osteoclastogenesis via down-regulation of NF-kappaB activity and suggest a new function for PPCA as an NF-kappaB-degrading enzyme in addition to its known multifunctional properties.

  19. Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

    Science.gov (United States)

    Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

    2009-10-15

    Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

  20. Obtenção e avaliação da proteína BYC (Boophilus Yolk pro-Cathepsin) recombinante em uma vacina contra o carrapato Boophilus microplus

    OpenAIRE

    Alexandre Trindade Leal

    2006-01-01

    O carrapato Boophilus microplus é um ectoparasita hematófago que infesta os rebanhos bovinos de regiões tropicais e subtropicais, causando grande prejuízo à pecuária. O principal método de controle deste parasita baseia-se no uso de acaricidas, entretanto, o uso de vacinas tem sido estudado como um método de controle promissor. A Boophilus Yolk pro-Cathepsin (BYC) é uma aspártico proteinase presente no ovo do carrapato e envolvida na embriogênese que foi anteriormente testada como imunógeno v...

  1. Histopathology of Anticarsia gemmatalis Hübner (Lepidoptera; Noctuidae treated with Nucleopolyhedrovirus and Bacillus thuringiensis serovar kurstaki Histopatologia de Anticarsia gemmatalis Hübner (Lepidoptera; Noctuidae tratadas com Virus de Poliedrose Nuclear e Bacillus thuringiensis sorovar kurstaki

    Directory of Open Access Journals (Sweden)

    Neiva Knaak

    2005-06-01

    Full Text Available The Anticarsia gemmatalis is responsible for the use of chemical insecticides in the soybean culture, causing a significant increase in the costs of farming and a great unbalance in the ecosystem. The use of microbial agents, like Bacillus thuringiensis serovar kurstaki (Btk and Anticarsia gemmatalis nucleopolyhedrovirus (AgNPV, they are an alternative to chemical control of the pest insects. In the interaction analysis of the entomopathogenic bacteria and virus it is considered important the in vitro action mode of these microbiology control agents. Therefore, the present study aims the histopathological analysis of the A. gemmatalis larvae digestive system after the interaction in vivo of the entomopathogenic Btk and AgNPV, represented the Dipel and Baculovirus anticarsia formulations, respectively. The evaluations were realized in larvae of 2nd instar, in which the mortality was evaluated daily, and a histopathology was done with collected larvae in time of 1, 3, 6, 12 and 24 hours after the treatments application. The results of the in vivo assays reveal that the treatment using the association of AgNPV-Btk (98.68% of mortality was more efficient than using AgNPV isolatedly (81.28% of mortality, but the Btk when used isolatedly had a mortality of 100%. The treatments showed significant (PA Anticarsia gemmatalis é responsável pelo uso de inseticidas químicos na cultura da soja, ocasionando um significativo aumento nos custos das lavouras e um grande desequilíbrio no ecossistema. O uso de agentes microbianos, como Bacillus thuringiensis sorovar kurstaki (Btk e Vírus de Poliedrose Nuclear de Anticarsia gemmatalis (VPNAg, é uma alternativa para o controle químico de insetos-praga. Na análise da interação de bactérias e vírus entomopatogênicos, considera-se importante o modo de ação in vitro desses agentes de controle microbiano. Assim, o presente trabalho objetiva a análise histopatológica do sistema digestivo das lagartas de A

  2. Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)*

    Science.gov (United States)

    Zhou, Nan; Pan, Ting; Zhang, Junsong; Li, Qianwen; Zhang, Xue; Bai, Chuan; Huang, Feng; Peng, Tao; Zhang, Jianhua; Liu, Chao; Tao, Liang; Zhang, Hui

    2016-01-01

    Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm. Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. PMID:26953343

  3. Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

    Science.gov (United States)

    Zhou, Nan; Pan, Ting; Zhang, Junsong; Li, Qianwen; Zhang, Xue; Bai, Chuan; Huang, Feng; Peng, Tao; Zhang, Jianhua; Liu, Chao; Tao, Liang; Zhang, Hui

    2016-04-22

    Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Cathepsin S Is Involved in Th17 Differentiation Through the Upregulation of IL-6 by Activating PAR-2 after Systemic Exposure to Lipopolysaccharide from Porphyromonas gingivalis

    Directory of Open Access Journals (Sweden)

    Masato Dekita

    2017-07-01

    Full Text Available Positive links have been found between periodontitis and numerous diseases in humans via persistent inflammation throughout the body. However, the main factors responsible for maintaining this pro-inflammatory condition are poorly understood. The spleen, the largest secondary immune organ, is a central hub regulating the immune response/inflammation due to the dendritic cell (DC response to CD4+ T cell subtype differentiation, and lysosomal proteinase cathepsin S (CatS is known to be involved in DC functions. In the present study, we found that CatS-induced IL-6 production by splenic DCs subsequently promotes Th17 differentiation, in response to systemic exposure to lipopolysaccharide derived from Porphyromonas gingivalis (PgLPS. The population of CD11c+ DCs was significantly increased in the splenic marginal zone (MZ locally of wild-type (DBA/2 mice with splenomegaly but not in that of CatS deficient (CatS-/- mice after systemic exposure to PgLPS for 7 consecutive days (5 mg/kg/day, intraperitoneal. Similarly, the population of Th17+CD4+ T cells was also significantly increased in the splenic MZ of wild-type mice but not in that of CatS-/- mice after PgLPS exposure. Furthermore, the increase in the Th17+ CD4+ T cell population paralleled increases in the levels of CatS and IL-6 in CD11c+ cells in the splenic MZ. In isolated primary splenic CD11c+ cells, the mRNA expression and the production of IL-6 was dramatically increased in wild-type mice but not in CatS-/- mice after direct stimulation with PgLPS (1 μg/ml, and this PgLPS-induced increase in the IL-6 expression was completely abolished by pre-treatment with Z-Phe-Leu-COCHO (Z-FL, the specific inhibitor of CatS. The PgLPS activated protease-activated receptor (PAR 2 in the isolated splenic CD11c+ cells was also significantly inhibited by CatS deficiently. In addition, the PgLPS-induced increase in the IL-6 production by splenic CD11c+ cells was completely abolished by pre-treatment with

  5. Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L.

    Science.gov (United States)

    Ehmke, Veronika; Winkler, Edwin; Banner, David W; Haap, Wolfgang; Schweizer, W Bernd; Rottmann, Matthias; Kaiser, Marcel; Freymond, Céline; Schirmeister, Tanja; Diederich, François

    2013-06-01

    The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition constants in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed

  6. Influence and interactions of cathepsin D, HLA-DRB1 and APOE on cognitive abilities in an older non-demented population.

    Science.gov (United States)

    Payton, A; van den Boogerd, E; Davidson, Y; Gibbons, L; Ollier, W; Rabbitt, P; Worthington, J; Horan, M; Pendleton, N

    2006-01-01

    Cathepsin D (CTSD), human leukocyte antigen DRB1 (HLA-DRB1) and apolipoprotein E (APOE) have all been associated with cognitive ability in both demented and non-demented individuals. CTSD is a pleiotrophic protein whose functions include the processing of proteins prior to presentation by HLA. Several studies have also reported that a functional exon 2 polymorphism in the CTSD gene interacts with APOEepsilon4 resulting in an increased risk of developing Alzheimer's disease (AD). We have previously reported that the CTSD exon 2 polymorphism regulates fluid intelligence. In this study, we extend this finding to other cognitive domains and investigate interactions with APOE and HLA-DRB1. Using a cohort of 766 non-demented volunteers, we found that the CTSD exon 2 T allele was associated with a decrease in several cognitive domains that comprise processing speed [random letters (RLs) test, P = 0.012; alphabet-coding task (ACT), P = 0.001], spatial recall (SR) (P = 0.016) and an additional test of fluid intelligence (P = 0.010). We also observed that the HLA-DR1 was associated with enhanced cumulative recall ability (P = 0.006), and conversely HLA-DR5 was associated with diminished delayed verbal recall and SR abilities (P = 0.014 and P = 0.003, respectively). When analysed independently, APOEepsilon4 did not influence any cognitive domains. In contrast, CTSD T/APOEepsilon4-positive volunteers scored lower on tests of fluid intelligence (P = 0.015), processing speed (ACT, P = 0.001; RL, P = 0.013) and immediate recall (P = 0.029). Scores were lower for all these tests than when CTSD and APOE were analysed independently. This supports previous findings in AD that have also reported an epistatic interaction. In addition, we found that CTSD T/HLA-DR2-positive volunteers had reduced processing speed (ACT, P = 0.040; RL, P = 0.014) and had significantly lower cumulative and SR abilities (P = 0.003 and P = 0.001, respectively). Biological interaction between these two

  7. Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble γ-glutamyl transpeptidase

    Directory of Open Access Journals (Sweden)

    Sajid Mohammed

    2006-05-01

    Full Text Available Background In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. Results Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a γ-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. γ-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG γ-glutamyl transpeptidase (GGT-1 is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong γ-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. Conclusion We have applied biochemical approaches, not used

  8. Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase.

    Science.gov (United States)

    Walker, Michael J; Rylett, Caroline M; Keen, Jeff N; Audsley, Neil; Sajid, Mohammed; Shirras, Alan D; Isaac, R Elwyn

    2006-05-02

    In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a gamma-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. gamma-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG gamma-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong gamma-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. We have applied biochemical approaches, not used previously, to characterise

  9. 2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the β-subunit of G-protein in human neutrophils.

    Science.gov (United States)

    Liao, Hsiang-Ruei; Chen, Ih-Sheng; Liu, Fu-Chao; Lin, Shinn-Zhi; Tseng, Ching-Ping

    2018-06-15

    This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox . Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Multiple Input - Multiple Output (MIMO) SAR

    Data.gov (United States)

    National Aeronautics and Space Administration — This effort will research and implement advanced Multiple-Input Multiple-Output (MIMO) Synthetic Aperture Radar (SAR) techniques which have the potential to improve...

  11. Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis.

    Science.gov (United States)

    Taniguchi, M; Ogiso, H; Takeuchi, T; Kitatani, K; Umehara, H; Okazaki, T

    2015-04-09

    We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM-ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM-ceramide-CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.

  12. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K; Constituintes quimicos das cascas do caule de Vochysia thyrsoidea Pohl. (Vochysiaceae) e avaliacao das atividades citotoxica e inibitoria frente as catepsinas B e K

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, Lorena Ramos Freitas de; Silva, Jame' s A. da; Vieira, Paulo Cezar [Universidade Federal de Sao Carlos (UFSCar), SP (Brazil). Departamento de Quimica; Costa, Maisa Borges; Santos, Mirley Luciene dos; Menezes, Antonio Carlos Severo, E-mail: amenezes@ueg.br [Universidade Estadual de Goias (UEG), Anapolis, GO (Brazil). Unidade Universitaria de Ciencias Exatas e Tecnologicas; Sbardelotto, Aline Borba; Pessoa, Claudia do O; Moraes, Manoel Odorico de [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil). Fac. de Medicina. Dept. de Fisiologia e Farmacologia

    2014-04-15

    A new flavonoid, catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC{sub 50} = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC{sub 50} = 36.80 μM and IC{sub 50} = 25.37 μM, respectively (author)

  13. Multiple-Ring Digital Communication Network

    Science.gov (United States)

    Kirkham, Harold

    1992-01-01

    Optical-fiber digital communication network to support data-acquisition and control functions of electric-power-distribution networks. Optical-fiber links of communication network follow power-distribution routes. Since fiber crosses open power switches, communication network includes multiple interconnected loops with occasional spurs. At each intersection node is needed. Nodes of communication network include power-distribution substations and power-controlling units. In addition to serving data acquisition and control functions, each node acts as repeater, passing on messages to next node(s). Multiple-ring communication network operates on new AbNET protocol and features fiber-optic communication.

  14. Generalized internal multiple imaging

    KAUST Repository

    Zuberi, Mohammad Akbar Hosain

    2014-12-04

    Various examples are provided for generalized internal multiple imaging (GIMI). In one example, among others, a method includes generating a higher order internal multiple image using a background Green\\'s function and rendering the higher order internal multiple image for presentation. In another example, a system includes a computing device and a generalized internal multiple imaging (GIMI) application executable in the computing device. The GIMI application includes logic that generates a higher order internal multiple image using a background Green\\'s function and logic that renders the higher order internal multiple image for display on a display device. In another example, a non-transitory computer readable medium has a program executable by processing circuitry that generates a higher order internal multiple image using a background Green\\'s function and renders the higher order internal multiple image for display on a display device.

  15. MultipleColposcopyJCO

    Science.gov (United States)

    Performing multiple biopsies during a procedure known as colposcopy—visual inspection of the cervix—is more effective than performing only a single biopsy of the worst-appearing area for detecting cervical cancer precursors. This multiple biopsy approach

  16. Salmonella Infections - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Salmonella Infections URL of this page: https://medlineplus.gov/ ... V W XYZ List of All Topics All Salmonella Infections - Multiple Languages To use the sharing features ...

  17. Health Literacy - Multiple Languages

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    ... Here: Home → Multiple Languages → All Health Topics → Health Literacy URL of this page: https://medlineplus.gov/languages/ ... W XYZ List of All Topics All Health Literacy - Multiple Languages To use the sharing features on ...

  18. Cosmetic Dentistry - Multiple Languages

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    ... Here: Home → Multiple Languages → All Health Topics → Cosmetic Dentistry URL of this page: https://medlineplus.gov/languages/ ... W XYZ List of All Topics All Cosmetic Dentistry - Multiple Languages To use the sharing features on ...

  19. Atrial Fibrillation - Multiple Languages

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    ... Are Here: Home → Multiple Languages → All Health Topics → Atrial Fibrillation URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Atrial Fibrillation - Multiple Languages To use the sharing features on ...

  20. Zika Virus - Multiple Languages

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    ... Are Here: Home → Multiple Languages → All Health Topics → Zika Virus URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Zika Virus - Multiple Languages To use the sharing features on ...

  1. Elder Abuse - Multiple Languages

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    ... Are Here: Home → Multiple Languages → All Health Topics → Elder Abuse URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Elder Abuse - Multiple Languages To use the sharing features on ...

  2. Herbal Medicine - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Herbal Medicine URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Herbal Medicine - Multiple Languages To use the sharing features on ...

  3. Generalized internal multiple imaging

    KAUST Repository

    Zuberi, Mohammad Akbar Hosain; Alkhalifah, Tariq

    2014-01-01

    Various examples are provided for generalized internal multiple imaging (GIMI). In one example, among others, a method includes generating a higher order internal multiple image using a background Green's function and rendering the higher order internal multiple image for presentation. In another example, a system includes a computing device and a generalized internal multiple imaging (GIMI) application executable in the computing device. The GIMI application includes logic that generates a higher order internal multiple image using a background Green's function and logic that renders the higher order internal multiple image for display on a display device. In another example, a non-transitory computer readable medium has a program executable by processing circuitry that generates a higher order internal multiple image using a background Green's function and renders the higher order internal multiple image for display on a display device.

  4. Domestic Violence - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Domestic Violence URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Domestic Violence - Multiple Languages To use the sharing features on ...

  5. Multiple Primary Tumors

    African Journals Online (AJOL)

    2017-12-05

    Dec 5, 2017 ... Multiple primary tumors occur in clinical practice causing diagnostic dilemma. It is not very .... was estrogen receptor negative, progesterone receptor negative, and ... cervical, ovarian, and urinary bladder cancers. Multiple.

  6. Diabetic Foot - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Diabetic Foot URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Diabetic Foot - Multiple Languages To use the sharing features on ...

  7. Child Abuse - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Child Abuse URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Child Abuse - Multiple Languages To use the sharing features on ...

  8. Fatigue and Multiple Sclerosis

    Science.gov (United States)

    ... to navigation Skip to content Menu Navigation National Multiple Sclerosis Society Sign In In Your Area Donate Donate ... of MS What Causes MS? Who Gets MS? Multiple Sclerosis FAQs Types of MS Related Conditions Symptoms & Diagnosis ...

  9. Rotavirus Infections - Multiple Languages

    Science.gov (United States)

    ... Are Here: Home → Multiple Languages → All Health Topics → Rotavirus Infections URL of this page: https://medlineplus.gov/ ... V W XYZ List of All Topics All Rotavirus Infections - Multiple Languages To use the sharing features ...

  10. Neutron Multiplicity Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Frame, Katherine Chiyoko [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-06-28

    Neutron multiplicity measurements are widely used for nondestructive assay (NDA) of special nuclear material (SNM). When combined with isotopic composition information, neutron multiplicity analysis can be used to estimate the spontaneous fission rate and leakage multiplication of SNM. When combined with isotopic information, the total mass of fissile material can also be determined. This presentation provides an overview of this technique.

  11. Neutron multiplication measurement instrument

    International Nuclear Information System (INIS)

    Nixon, K.V.; Dowdy, E.J.; France, S.W.; Millegan, D.R.; Robba, A.A.

    1983-01-01

    The Advanced Nuclear Technology Group of the Los Alamos National Laboratory is now using intelligent data-acquisition and analysis instrumentation for determining the multiplication of nuclear material. Earlier instrumentation, such as the large NIM-crate systems, depended on house power and required additional computation to determine multiplication or to estimate error. The portable, battery-powered multiplication measurement unit, with advanced computational power, acquires data, calculates multiplication, and completes error analysis automatically. Thus, the multiplication is determined easily and an available error estimate enables the user to judge the significance of results

  12. Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways.

    Science.gov (United States)

    Pranjol, Md Zahidul I; Gutowski, Nicholas J; Hannemann, Michael; Whatmore, Jacqueline L

    2018-01-01

    Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Progranulin acts as a shared chaperone and regulates multiple lysosomal enzymes

    Directory of Open Access Journals (Sweden)

    Jinlong Jian

    2017-09-01

    Full Text Available Multifunctional factor progranulin (PGRN plays an important role in lysosomes, and its mutations and insufficiency are associated with lysosomal storage diseases, including neuronal ceroid lipofuscinosis and Gaucher disease (GD. The first breakthrough in understanding the molecular mechanisms of PGRN as regulator of lysosomal storage diseases came unexpectedly while investigating the role of PGRN in inflammation. Challenged PGRN null mice displayed typical features of GD. In addition, GRN gene variants were identified in GD patients and the serum levels of PGRN were significantly lower in GD patients. PGRN directly binds to and functions as a chaperone of the lysosomal enzyme β-glucocerebrosidase (GCaase, whose mutations cause GD. In addition, its C-terminus containing granulin E domain, termed Pcgin (PGRN C-terminus for GCase Interaction, is required for the association between PGRN and GCase. The concept that PGRN acts as a chaperone of lysosomal enzymes was further supported and extended by a recent article showing that PGRN acts as a chaperone molecule of lysosomal enzyme cathepsin D (CSTD, and the association between PGRN and CSTD is also mediated by PGRN's C-terminal granulin E domain. Collectively, these reports suggest that PGRN may act as a shared chaperone and regulates multiple lysosomal enzymes.

  14. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    Science.gov (United States)

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  15. Dendrimer-magnetic nanoparticles as multiple stimuli responsive and enzymatic drug delivery vehicle

    International Nuclear Information System (INIS)

    Chandra, Sudeshna; Noronha, Glen; Dietrich, Sascha; Lang, Heinrich; Bahadur, Dhirendra

    2015-01-01

    Two different chain lengths of (poly)ethylene glycol-PAMAM dendrimers namely, L6-PEG-PAMAM and S6-PEG-PAMAM with six end-grafted ethylene glycol ether-tentacles of type CH 2 CH 2 C(O)O(CH 2 CH 2 O) 9 CH 3 and CH 2 CH 2 C(O)O(CH 2 CH 2 O) 2 C 2 H 5 , respectively, were synthesized. These dendrimers have multiple σ-donor capabilities and therefore, were used for stabilizing the magnetite (Fe 3 O 4 ) nanoparticles. Both the dendrimer-magnetic nanoparticles (L6-PEG-PAMAM-MNPs and S6-PEG-PAMAM-MNPs) were characterized by different spectroscopic and microstructural techniques. The nanoparticles were mesoporous and superparamagnetic and therefore, explored for their possible use in delivery of cancer drug, doxorubicin (DOX). In the developed drug delivery system, achieving high drug-loading efficiency with controllable release were the main challenges. The change in zeta potential and quenching of fluorescence intensity suggests chemical interaction between DOX and the nanoparticles. The loading efficiency was calculated to be over 95% with a sustained pH and temperature sensitive release. Further, enzyme cathepsin B has also been used to degrade the dendritic shell to trigger sustained drug release in the vicinity of tumor cells

  16. Dendrimer-magnetic nanoparticles as multiple stimuli responsive and enzymatic drug delivery vehicle

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Sudeshna; Noronha, Glen [Metallurgical and Materials Science Department, Indian Institute of Technology Bombay, Powai, Mumbai, 400076 (India); Dietrich, Sascha; Lang, Heinrich [Technische Universität Chemnitz, Institute of Chemistry, Straße der Nationen 62, d-09111 Chemnitz (Germany); Bahadur, Dhirendra, E-mail: dhirenb@iitb.ac.in [Metallurgical and Materials Science Department, Indian Institute of Technology Bombay, Powai, Mumbai, 400076 (India)

    2015-04-15

    Two different chain lengths of (poly)ethylene glycol-PAMAM dendrimers namely, L6-PEG-PAMAM and S6-PEG-PAMAM with six end-grafted ethylene glycol ether-tentacles of type CH{sub 2}CH{sub 2}C(O)O(CH{sub 2}CH{sub 2}O){sub 9}CH{sub 3} and CH{sub 2}CH{sub 2}C(O)O(CH{sub 2}CH{sub 2}O){sub 2}C{sub 2}H{sub 5}, respectively, were synthesized. These dendrimers have multiple σ-donor capabilities and therefore, were used for stabilizing the magnetite (Fe{sub 3}O{sub 4}) nanoparticles. Both the dendrimer-magnetic nanoparticles (L6-PEG-PAMAM-MNPs and S6-PEG-PAMAM-MNPs) were characterized by different spectroscopic and microstructural techniques. The nanoparticles were mesoporous and superparamagnetic and therefore, explored for their possible use in delivery of cancer drug, doxorubicin (DOX). In the developed drug delivery system, achieving high drug-loading efficiency with controllable release were the main challenges. The change in zeta potential and quenching of fluorescence intensity suggests chemical interaction between DOX and the nanoparticles. The loading efficiency was calculated to be over 95% with a sustained pH and temperature sensitive release. Further, enzyme cathepsin B has also been used to degrade the dendritic shell to trigger sustained drug release in the vicinity of tumor cells.

  17. Accurate scaling on multiplicity

    International Nuclear Information System (INIS)

    Golokhvastov, A.I.

    1989-01-01

    The commonly used formula of KNO scaling P n =Ψ(n/ ) for descrete distributions (multiplicity distributions) is shown to contradict mathematically the condition ΣP n =1. The effect is essential even at ISR energies. A consistent generalization of the concept of similarity for multiplicity distributions is obtained. The multiplicity distributions of negative particles in PP and also e + e - inelastic interactions are similar over the whole studied energy range. Collider data are discussed. 14 refs.; 8 figs

  18. ALICE Photon Multiplicity Detector

    CERN Multimedia

    Nayak, T

    2013-01-01

    Photon Multiplicity Detector (PMD) measures the multiplicity and spatial distribution of photons in the forward region of ALICE on a event-by-event basis. PMD is a pre-shower detector having fine granularity and full azimuthal coverage in the pseudo-rapidity region 2.3 < η < 3.9.

  19. Body Weight - Multiple Languages

    Science.gov (United States)

    ... Supplements Videos & Tools You Are Here: Home → Multiple Languages → All Health Topics → Body Weight URL of this page: https://medlineplus.gov/ ... V W XYZ List of All Topics All Body Weight - Multiple Languages To use the sharing features on this page, ...

  20. Multiplicity in difference geometry

    OpenAIRE

    Tomasic, Ivan

    2011-01-01

    We prove a first principle of preservation of multiplicity in difference geometry, paving the way for the development of a more general intersection theory. In particular, the fibres of a \\sigma-finite morphism between difference curves are all of the same size, when counted with correct multiplicities.

  1. Applying Multiple Intelligences

    Science.gov (United States)

    Christodoulou, Joanna A.

    2009-01-01

    The ideas of multiple intelligences introduced by Howard Gardner of Harvard University more than 25 years ago have taken form in many ways, both in schools and in other sometimes-surprising settings. The silver anniversary of Gardner's learning theory provides an opportunity to reflect on the ways multiple intelligences theory has taken form and…

  2. Formation of multiple networks

    DEFF Research Database (Denmark)

    Magnani, Matteo; Rossi, Luca

    2013-01-01

    we introduce the first network formation model for multiple networks. Network formation models are among the most popular tools in traditional network studies, because of both their practical and theoretical impact. However, existing models are not sufficient to describe the generation of multiple...

  3. Orchestrating Multiple Intelligences

    Science.gov (United States)

    Moran, Seana; Kornhaber, Mindy; Gardner, Howard

    2006-01-01

    Education policymakers often go astray when they attempt to integrate multiple intelligences theory into schools, according to the originator of the theory, Howard Gardner, and his colleagues. The greatest potential of a multiple intelligences approach to education grows from the concept of a profile of intelligences. Each learner's intelligence…

  4. Assessing Children's Multiplicative Thinking

    Science.gov (United States)

    Hurst, Chris; Hurrell, Derek

    2016-01-01

    Multiplicative thinking is a "big idea" of mathematics that underpins much of the mathematics learned beyond the early primary school years. This paper reports on a current study that utilises an interview tool and a written quiz to gather data about children's multiplicative thinking. The development of the tools and some of the…

  5. Insecticidal Efficacy of Azadirachta indica, Nucleopolyhedrovirus and Chlorantraniliprole Singly or Combined against Field Populations fo Helicoverpa armigera Hübner (Lepidoptera: Noctuidae Eficacia Insecticida de Azadirachta indica, Nucleopolihedrovirus y Clorantraniliprol solo y sus Aplicaciones Integradas contra Poblaciones de Campo de Helicoverpa armigera Hübner (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Waqas Wakil

    2012-03-01

    Full Text Available The development of resistance in cosmopolitan insect Helicoverpa armigera Hubner (Lepidoptera: Noctuidae forced the researchers for alternative control measures. In the present study, insecticidal efficacy of formulations of Azadirachta indica, a Nucleopolyhedrovirus (NPV, and new anthranilic diamide insecticide (chlorantraniliprole formulations was determined against 2nd, through 5th larval instars of H. armigera collected from diverse geographical locations in the Punjab province, Pakistan. Azadirachta indica was applied at 5 μL L-1; NPV at 2.1 x 10(5 polyhedral occlusion bodies (POB mL4 and chlorantraniliprole at 0.01 μL L-1, either alone or in combinations with each other. The bioassays were conducted at 27 ± 1 °C and 65 ± 5% relative humidity. The mortality varied greatly among treatments, larval instars, and locations. The combinations of NPV with A. indica and chlorantraniliprole caused higher mortality, pupation and produced an additive effect compared to their application singly in all the tested populations. The population from Rawalpindi was always susceptible while the Gujranwala was the resistant. The results herein suggest that the effectiveness of NPV and A. indica can be improved by the presence of chlorantraniliprole against the larvae of H. armigera.Se determinó la eficacia insecticida de formulaciones de Azadirachta indica, Nucleopolihedrovirus (VPN y el nuevo insecticida diamida antranílico (clorantraniliprol en contra de segundo, tercero, cuarto y quinto estadios larvales de Helicoverpa armigera Hubner (Lepidoptera: Noctuidae recogidos de diversas ubicaciones geográficas de la provincia de Punjab, Pakistán. Azadirachta indica se aplicó en dosis de 5 μL L-1; VPN en dosis 2.1 x 10(5 POB mL-1 y clorantraniliprol fue 0,01 μL L-1 ya sea solos o en combinaciones. Los bioensayos se realizaron a 27 ± 1 °C y 65 ± 5% de humedad relativa. La mortalidad fue notablemente variada entre los tratamientos, estadios larvales y

  6. Safe Dynamic Multiple Inheritance

    DEFF Research Database (Denmark)

    Ernst, Erik

    2002-01-01

    Multiple inheritance and similar mechanisms are usually only supported at compile time in statically typed languages. Nevertheless, dynamic multiple inheritance would be very useful in the development of complex systems, because it allows the creation of many related classes without an explosion...... in the size and level of redundancy in the source code. In fact, dynamic multiple inheritance is already available. The language gbeta is statically typed and has supported run-time combination of classes and methods since 1997, by means of the combination operator '&'. However, with certain combinations...

  7. A multiplicity logic unit

    International Nuclear Information System (INIS)

    Bialkowski, J.; Moszynski, M.; Zagorski, A.

    1981-01-01

    The logic diagram principle of operation and some details of the design of the multiplicity logic unit are presented. This unit was specially designed to fulfil the requirements of a multidetector arrangement for gamma-ray multiplicity measurements. The unit is equipped with 16 inputs controlled by a common coincidence gate. It delivers a linear output pulse with the height proportional to the multiplicity of coincidences and logic pulses corresponding to 0, 1, ... up to >= 5-fold coincidences. These last outputs are used to steer the routing unit working with the multichannel analyser. (orig.)

  8. Fission neutron multiplicity calculations

    International Nuclear Information System (INIS)

    Maerten, H.; Ruben, A.; Seeliger, D.

    1991-01-01

    A model for calculating neutron multiplicities in nuclear fission is presented. It is based on the solution of the energy partition problem as function of mass asymmetry within a phenomenological approach including temperature-dependent microscopic energies. Nuclear structure effects on fragment de-excitation, which influence neutron multiplicities, are discussed. Temperature effects on microscopic energy play an important role in induced fission reactions. Calculated results are presented for various fission reactions induced by neutrons. Data cover the incident energy range 0-20 MeV, i.e. multiple chance fission is considered. (author). 28 refs, 13 figs

  9. The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

    Science.gov (United States)

    Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

    2015-01-01

    In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

  10. Multiple sclerosis research

    International Nuclear Information System (INIS)

    Battaglia, M.A.

    1990-01-01

    This volume proceedings contains four contributions which are in INIS scope, dealing with MRI and SPECT in the diagnosis of multiple sclerosis and assessment of disease activity. (H.W.). refs.; figs.; tabs

  11. Generalized internal multiple imaging

    KAUST Repository

    Zuberi, M. A. H.; Alkhalifah, Tariq Ali

    2014-01-01

    Internal multiples deteriorate the image when the imaging procedure assumes only single scattering, especially if the velocity model does not have sharp contrasts to reproduce such scattering in the Green’s function through forward modeling

  12. Connecting the Production Multiple

    DEFF Research Database (Denmark)

    Lichen, Alex Yu; Mouritsen, Jan

    &OP process itself is a fluid object, but there is still possibility to organise the messy Production. There are connections between the Production multiple and the managerial technology fluid. The fluid enacted the multiplicity of Production thus making it more difficult to be organised because there were...... in opposite directions. They are all part of the fluid object. There is no single chain of circulating references that makes the object a matter of fact. Accounting fluidity means that references drift back and forth and enact new realities also connected to the chain. In this setting future research may......This paper is about objects. It follows post ANT trajectories and finds that objects are multiple and fluid. Extant classic ANT inspired accounting research largely sees accounting inscriptions as immutable mobiles. Although multiplicity of objects upon which accounting acts has been explored...

  13. Multiple sex partner

    African Journals Online (AJOL)

    User

    intercourse, about 60% reported having a single sexual partner and 40% reported having multiple ... masturbation, start having sex at a younger age, have sex with married people and/or .... sex were considered unacceptable by 89 vs.

  14. Multiple Stages 2

    DEFF Research Database (Denmark)

    Andreasen, John

    Multiple stages 2: theatrical futures, set design, community plays, cultural capitals, democracy & drama, WWII dramas, performance on adoption, promenade about emigration, qualities in political theatre, performance analysis, dramaturgical education, Toulmin Variations...

  15. Rehabilitation and multiple sclerosis

    DEFF Research Database (Denmark)

    Dalgas, Ulrik

    2011-01-01

    In a chronic and disabling disease like multiple sclerosis, rehabilitation becomes of major importance in the preservation of physical, psychological and social functioning. Approximately 80% of patients have multiple sclerosis for more than 35 years and most will develop disability at some point......, a paradigm shift is taking place and it is now increasingly acknowledged that exercise therapy is both safe and beneficial. Robot-assisted training is also attracting attention in multiple sclerosis rehabilitation. Several sophisticated commercial robots exist, but so far the number of scientific studies...... promising. This drug has been shown to improve walking ability in some patients with multiple sclerosis, associated with a reduction of patients' self-reported ambulatory disability. Rehabilitation strategies involving these different approaches, or combinations of them, may be of great use in improving...

  16. Generalized internal multiple imaging

    KAUST Repository

    Zuberi, M. A. H.

    2014-08-05

    Internal multiples deteriorate the image when the imaging procedure assumes only single scattering, especially if the velocity model does not have sharp contrasts to