Browsing by Subject "INHIBITORS"

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  • Lai, Jeffrey K. F.; Sam, I-Ching; Verlhac, Pauline; Baguet, Joel; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun (2017)
    Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.
  • Aly, Ashraf A.; El-Emary, Talaat; Mourad, Aboul-Fetouh E.; Alyan, Zainab Khallaf; Bräse, Stefan; Nieger, Martin (2019)
    5-Carbohydrazides and 5-carbonylazides of pyrazolo[3,4-b]pyridines are used to synthesize new heterocyclic derivatives. Some unexpected behaviors are observed in the reactions of the above two species. The structures of the obtained compounds are proved by spectroscopic studies together with elemental and X-ray structure analyses.
  • Wannasarit, Saowanee; Wang, Shiqi; Figueiredo, Patricia; Trujillo Olvera, Claudia Ximenia; Eburnea, Francesca; Simón-Gracia, Lorena; Correia, Alexandra; Ding, Yaping; Teesalu, Tambet; Liu, Dongfei; Wiwattanapatapee, Ruedeekorn; Santos, Hélder A.; Li, Wei (2019)
    Achieving cellular internalization and endosomal escape remains a major challenge for many antitumor therapeutics, especially macromolecular drugs. Viral drug carriers are reported for efficient intracellular delivery, but with limited choices of payloads. In this study, a novel polymeric nanoparticle (ADMAP) is developed, resembling the structure and functional features of a virus. ADMAP is synthesized by grafting endosomolytic poly(lauryl methacrylate‐co‐methacrylic acid) on acetalated dextran. The endosomolytic polymer mimics the capsid protein for endosomal escape, and acetalated dextran resembles the viral core for accommodating payloads. After polymer synthesis, the subsequent controlled nanoprecipitation on a microfluidic device yields uniform nanoparticles with high encapsulation efficiency. At late endosomal pH (5.0), the ADMAP particles successfully destabilize endosomal membranes and release the drug payloads synergistically, resulting in a greater therapeutic efficacy compared with that of free anticancer drugs. Further conjugation of a tumor‐penetrating peptide enhances the antitumor efficacy toward 3D spheroids and finally leads to spheroid disintegration. The unique structure along with the synergistic endosomal escape and drug release make ADMAP nanoparticles favorable for intracellular delivery of antitumor therapeutics.
  • Singh, Abhishek A.; Mandoli, Amit; Prange, Koen H. M.; Laakso, Marko; Martens, Joost H. A. (2017)
    Chromosomal translocations are one of the hallmarks of acute myeloid leukemia (AML), often leading to gene fusions and expression of an oncofusion protein. Over recent years it has become clear that most of the AML associated oncofusion proteins molecularly adopt distinct mechanisms for inducing leukemogenesis. Still these unique molecular properties of the chimeric proteins converge and give rise to a common pathogenic molecular mechanism. In the present study we compared genome-wide DNA binding and transcriptome data associated with AML1-ETO, CBFB-MYH11 and PML-RARA oncofusion protein expression to identify unique and common features. Our analyses revealed targeting of oncofusion binding sites to RUNX1 and ETS-factor occupied genomic regions. In addition, it revealed a highly comparable global histone acetylation pattern, similar expression of common target genes and related enrichment of several biological pathways critical for maintenance of AML, suggesting oncofusion proteins deregulate common gene programs despite their distinct binding signatures and mechanisms of action.
  • Jokela, Jouni; Oftedal, Linn; Herfindal, Lars; Permi, Perttu; Wahlsten, Matti; Doskeland, Stein Ove; Sivonen, Kaarina (2012)
  • Rautiola, Juhana; Lampinen, Anita; Mirtti, Tuomas; Ristimaki, Ari; Joensuu, Heikki; Bono, Petri; Saharinen, Pipsa (2016)
    The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.
  • Leino, Teppo O.; Turku, Ainoleena; Yli-Kauhaluoma, Jari Tapani; Kukkonen, Jyrki P.; Xhaard, Henri; Wallén, Erik A. A. (2018)
    A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX2 receptor. Based on the results, a series of azulene derivatives was synthesized and the binding to and activation of both orexin receptor subtypes were assessed. Two most promising binders were determined to have inhibition constants in the 3-9 mu M range and two other compounds showed weak OX2 receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX1 but not the OX2 receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold. (C) 2018 Elsevier Masson SAS. All rights reserved.
  • Rausalu, Kai; Utt, Age; Quirin, Tania; Varghese, Finny S.; Zusinaite, Eva; Das, Pratyush Kumar; Ahola, Tero; Merits, Andres (2016)
    Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.
  • Hedenbjork-Lager, Anders; Hamberg, Kristina; Paakkonen, Virve; Tjaderhane, Leo; Ericson, Dan (2016)
    Objective: Dental caries is a process driven by acids produced by oral microorganisms followed by degradation of the dentine collagen matrix by proteolytic enzymes. Matrix metalloproteinases (MMPs) have been suggested to contribute to caries by degrading collagen. The aim of this study was to develop a method for generating demineralized dentine matrix substrate (DDM) maintaining MMP-8 bioactivity and no interference with later assays. Such a substrate would allow study of the effects of various treatments on MMP-8 activity and collagen degradation in demineralized dentine. Design: Human dentine was powderized in a tissue grinder and frozen (-80 degrees C). The powder was demineralized in dialysis tubes, using EDTA or acetic acid. The demineralized dentine matrix (DDM) was harvested and analyzed for collagen content using SDS-PAGE. The DDM was subsequently suspended in PBS or TESCA buffer. Protein, MMP-8 (ELISA) and collagen (HYP) was analyzed directly or after 1 wk. Results: EDTA or acid demineralization of dentine using dialysis yielded a substrate rich in collagen coupled with preserved MMP-8 activity. Collagen degraded in room temperature, assessed by higher HYP amounts in the soluble fraction of DDM after one wk, indicating that the methods used preserved active DDM-components after the demineralization process. Conclusions: The presented demineralization methods both provided insoluble DDM substrates suitable for further intervention studies. However, it was found that the substrates differed depending on the demineralization method and buffers used. This needs further study to find an optimal technique for generating DDM with retained proteins as well as enzymatic bioactivity. (C) 2016 Elsevier Ltd. All rights reserved.
  • Sveen, Anita; Bruun, Jarle; Eide, Peter W.; Eilertsen, Ina A.; Ramirez, Lorena; Murumägi, Astrid; Arjama, Mariliina; Danielsen, Stine A.; Kryeziu, Kushtrim; Elez, Elena; Tabernero, Josep; Guinney, Justin; Palmer, Hector G.; Nesbakken, Arild; Kallioniemi, Olli; Dienstmann, Rodrigo; Lothe, Ragnhild A. (2018)
    Purpose: Response to standard oncologic treatment is limited in colorectal cancer. The gene expression-based consensus molecular subtypes (CMS) provide a new paradigm for stratified treatment and drug repurposing; however, drug discovery is currently limited by the lack of translation of CMS to preclinical models. Experimental Design: We analyzed CMS in primary colorectal cancers, cell lines, and patient-derived xenografts (PDX). For classification of preclinical models, we developed an optimized classifier enriched for cancer cell-intrinsic gene expression signals, and performed high-throughput in vitro drug screening (n = 459 drugs) to analyze subtype-specific drug sensitivities. Results: The distinct molecular and clinicopathologic characteristics of each CMS group were validated in a single-hospital series of 409 primary colorectal cancers. The new, cancer cell-adapted classifier was found to perform well in primary tumors, and applied to a panel of 148 cell lines and 32 PDXs, these colorectal cancer models were shown to recapitulate the biology of the CMS groups. Drug screening of 33 cell lines demonstrated subtype-dependent response profiles, confirming strong response to EGFR and HER2 inhibitors in the CMS2 epithelial/canonical group, and revealing strong sensitivity to HSP90 inhibitors in cells with the CMS1 microsatellite instability/immune and CMS4 mesenchymal phenotypes. This association was validated in vitro in additional CMS-predicted cell lines. Combination treatment with 5-fluorouracil and luminespib showed potential to alleviate chemoresistance in a CMS4 PDX model, an effect not seen in a chemosensitive CMS2 PDX model. Conclusions: We provide translation of CMS classification to preclinical models and uncover a potential for targeted treatment repurposing in the chemoresistant CMS4 group. (C) 2017 AACR.
  • Jones, Martin R.; Pinto, Ernani; Torres, Mariana A.; Dörr, Fabiane; Mazur-Marzec, Hanna; Szubert, Karolina; Tartaglione, Luciana; Dell'Aversano, Carmela; Miles, Christopher O.; Beach, Daniel G.; McCarron, Pearse; Sivonen, Kaarina; Fewer, David P.; Jokela, Jouni; Janssen, Elisabeth M.-L. (2021)
    Harmful cyanobacterial blooms, which frequently contain toxic secondary metabolites, are reported in aquatic environments around the world. More than two thousand cyanobacterial secondary metabolites have been reported from diverse sources over the past fifty years. A comprehensive, publically-accessible database detailing these secondary metabolites would facilitate research into their occurrence, functions and toxicological risks. To address this need we created CyanoMetDB, a highly curated, flat-file, openly-accessible database of cyanobacterial secondary metabolites collated from 850 peer-reviewed articles published between 1967 and 2020. CyanoMetDB contains 2010 cyanobacterial metabolites and 99 structurally related compounds. This has nearly doubled the number of entries with complete literature metadata and structural composition information compared to previously available open access databases. The dataset includes microcytsins, cyanopeptolins, other depsipeptides, anabaenopeptins, microginins, aeruginosins, cyclamides, cryptophycins, saxitoxins, spumigins, microviridins, and anatoxins among other metabolite classes. A comprehensive database dedicated to cyanobacterial secondary metabolites facilitates: (1) the detection and dereplication of known cyanobacterial toxins and secondary metabolites; (2) the identification of novel natural products from cyanobacteria; (3) research on biosynthesis of cyanobacterial secondary metabolites, including substructure searches; and (4) the investigation of their abundance, persistence, and toxicity in natural environments.
  • Salonen, Päivi H.; Salonen, Juha H.; Säilä, Hanna; Helminen, Mika; Linna, Miika; Kauppi, Markku J. (2020)
    OBJECTIVES: Children with juvenile idiopathic arthritis (JIA) may be predisposed to serious pneumonia due to modern disease-modifying anti-rheumatic treatment. In this nationwide retrospective study with clinical data, we describe the pneumonia episodes among children with JIA. METHODS: Patients under 18 years of age with JIA and pneumonia during 1998-2014 were identified in the National Hospital Discharge Register in Finland. Each individual patient record was reviewed, and detailed data on patients with JIA and pneumonia were retrieved, recorded, and analyzed. If the patient was hospitalized or received intravenous antibiotics, the pneumonia was considered serious. RESULTS: There were 157 episodes of pneumonia among 140 children with JIA; 111 episodes (71%) were serious (80% in 1998-2006 and 66% in 2007-2014). The mean age of the patients was 9 years. Forty-eight percent had active JIA and 46% had comorbidities. Disease-modifying anti-rheumatic drugs (DMARD) were used at the time of 135 episodes (86%): methotrexate (MTX) by 62% and biologic DMARDs (bDMARD) by 30%. There was no significant difference in the use of bDMARDs, MTX and glucocorticoids between the patient groups with serious and non-serious pneumonia episodes. During six of the episodes, intensive care was needed. Two patients (1.3%) died, the remaining ones recovered fully. CONCLUSIONS: Although the incidence of pneumonia and the use of immunosuppressive treatment among children with JIA increased from 1998 to 2014, the proportion of serious pneumonias in these patients decreased. There was no significant difference in the use of anti-rheumatic medication between patients with serious and non-serious pneumonia.Key Points• The incidence of serious pneumonias decreased from 1998 to 2014 among children with juvenile idiopathic arthritis (JIA).• There was no significant difference in the use of the disease-modifying anti-rheumatic medication between JIA patients with serious and non-serious pneumonias.• Active JIA, comorbidities, and combination medication were associated with nearly half of the pneumonias.
  • Narvi, Elli; Vaparanta, Katri; Karrila, Anna; Chakroborty, Deepankar; Knuutila, Sakari; Pulliainen, Arto; Sundvall, Maria; Elenius, Klaus (2018)
    Therapeutic protocols including EGFR antibodies in the context of oxaliplatin-based regimens have variable clinical effect in colorectal cancer. Here, we tested the effect of the EGFR antibody cetuximab in different sequential combinations with oxaliplatin on the growth of colorectal cancer cells in vitro and in vivo. Cetuximab reduced the efficacy of oxaliplatin when administered before oxaliplatin but provided additive effect when administered after oxaliplatin regardless of the KRAS or BRAF mutation status of the cells. Systemic gene expression and protein phosphorylation screens revealed alternatively activated pathways regulating apoptosis, cell cycle and DNA damage response. Functional assays indicated that cetuximab-induced arrest of the cells into the G1 phase of the cell cycle was associated with reduced responsiveness of the cells to subsequent treatment with oxaliplatin. In contrast, oxaliplatin-enhanced responsiveness to subsequent treatment with cetuximab was associated with increased apoptosis, inhibition of STAT3 activity and increased EGFR down-regulation. This preclinical study indicates that optimizing the sequence of administration may enhance the antitumor effect of combination therapy with EGFR antibodies and oxaliplatin.
  • Chen, Jason; Verni, Christopher C.; Jouppila, Annukka; Lassila, Riitta; Diamond, Scott L. (2018)
    Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s(-1)). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 mu g/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s(-1)) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.
  • BEEHIVE Collaboration; Wymant, Chris; Blanquart, Francois; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J.; Hall, Matthew; Hillebregt, Mariska; Ong, Swee Hoe; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M. Kate; Gunsenheimer-Bartmeyer, Barbara; Gunthard, Huldrych F.; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Berkhout, Ben; Cornelissen, Marion; Kellam, Paul; Reiss, Peter; Fraser, Christophe (2018)
    Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between-and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.
  • Aaltonen, Niina; Singha, Prosanta K.; Jakupovic, Hermina; Wirth, Thomas; Samaranayake, Haritha; Pasonen-Seppanen, Sanna; Rilla, Kirsi; Varjosalo, Markku; Edgington-Mitchell, Laura E.; Kasperkiewicz, Paulina; Drag, Marcin; Kälvälä, Sara; Moisio, Eemeli; Savinainen, Juha R.; Laitinen, Jarmo T. (2020)
    Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.
  • Raivola, Juuli; Hammaren, Henrik M.; Virtanen, Anniina T.; Bulleeraz, Vilasha; Ward, Alister C.; Silvennoinen, Olli (2018)
    Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (yc) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known. The pseudokinase domain (JAK homology 2, JH2) of JAK3 is of particular interest as approximately half of clinical JAK3 mutations cluster into it. In this study, we investigated the role of JH2s of JAK1 and JAK3 in IL-2R signaling and show that STAT5 activation requires both JH1 and JH2 of JAK1, while both JH1 and JH2 in JAK3 are specifically required for the cytokine-induction of cellular signaling. Characterization of recombinant JAK3 JH2 in thermal shift assay shows an unstable protein domain, which is strongly stabilized by ATP binding. Unexpectedly, nucleotide binding to JAK3 JH2 was found to be cation-independent. JAK3 JH2 showed higher nucleotide binding affinity in MANT-ATP and fluorescent polarization competition assays compared to the other JAK JH2s. Analysis of the functional role of ATP binding in JAK3 JH2 in cells and in zebrafish showed that disruption of ATP binding suppresses ligand-independent activation of clinical JAK3 gain-of-function mutations residing in either JH2 or JH1 but does not inhibit constitutive activation of oncogenic JAK1. ATP-binding site mutations in JAK3 JH2 do not, however, abrogate normal IL-2 signaling making them distinct from JH2 deletion or kinase-deficient JAK3. These findings underline the importance of JAK3 JH2 for cellular signaling in both ligand-dependent and in gain-of-function mutation-induced activation. Furthermore, they identify the JH2 ATP-binding site as a key regulatory region for oncogenic JAK3 signaling, and thus a potential target for therapeutic modulation.
  • Hanifeh, Mohsen; Rajamäki, Minna Marjaana; Syrjä, Pernilla; Mäkitalo, Laura; Kilpinen, Susanne; Spillmann, Thomas (2018)
    Background: Matrix metalloproteinases (MMPs) 2 and 9 are zinc-and calcium-dependent endopeptidases involved in the breakdown and reconstitution of extracellular matrix under both physiological and pathological conditions. Mucosal MMP-2 and -9 activities have been reported to be upregulated in the intestine of humans with inflammatory bowel disease (IBD), and in animal models of IBD. However, their involvement in the pathogenesis of canine chronic enteropathies (CE) is unknown. This study investigated mucosal pro-and active MMP-2 and -9 activities in dogs with CE and healthy dogs using gelatin zymography, and also to determine the association of their activities in dogs with CE with the canine IBD activity index (CIBDAI), histopathologic findings, the clinical outcome, and hypoalbuminemia. Intestinal mucosal samples from duodenum, ileum, colon, and cecum were collected from 40 dogs with CE and 18 healthy Beagle dogs. Results: In dogs with CE, the number of samples positive for mucosal pro-and active MMP-2 was significantly higher in the duodenum (P <0.0001 and P = 0.011, respectively), ileum (P = 0.002 and P = 0.018, respectively), and colon (P <0.0001 and P = 0.002, respectively), compared with healthy controls. Mucosal pro-MMP-9-positive samples in the duodenum and colon were significantly more frequent in dogs with CE than in healthy dogs (P = 0.0004 and P = 0.001, respectively). Despite the presence of mucosal samples positive for active MMP-9 in the intestinal segments of dogs with CE, the difference compared to healthy controls did not reach statistical significance. None of the intestinal mucosal samples in healthy dogs showed gelatinolytic activity corresponding to the control bands of active MMP-2 and -9. Mucosal active MMP-9 activities displayed a significant positive association with the severity of neutrophil infiltration in the duodenum (P = 00.040), eosinophils in the cecum (P = 00.037), and the CIBDAI score for ileum samples (P = 0.023). There was no significant association of pro-and active MMP-2 and -9 levels with the clinical outcome or hypoalbuminemia. Conclusions: This study is the first to demonstrate upregulation of mucosal pro-and active MMP-2 and pro-MMP-9 in the intestine of dogs with CE compared to healthy dogs. The results provide supporting evidence for the possible involvement of MMP-2 and -9 in the pathogenesis of canine CE.
  • Vorkapic, Emina; Dugic, Elma; Vikingsson, Svante; Roy, Joy; Mäyränpää, Mikko; Eriksson, Per; Wagsater, Dick (2016)
    Background: Abdominal aortic aneurysm (AAA) is characterized by vascular remodeling with increased infiltration of inflammatory cells and apoptosis/modulation of vascular smooth muscle cells (SMCs). Imatinib is a selective inhibitor of several tyrosine kinases, including PDGF receptors, Abl, and c-kit. The objective of this study was to characterize the potential protective role of imatinib on AAA development and the molecular mechanisms involved. Methods: Male ApoE(-/)-mice were infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Daily treatment with 10 mg/kg imatinib, or tap water as control, was provided via gavage for 4 weeks. Results: Treatment with imatinib was found to decrease the aortic diameter and vessel wall thickness, mediated by multiple effects. Imatinib treatment in AngII infused mice resulted in a reduced cellular infiltration of CD3 epsilon positive T lymphocytes by 86% and reduced gene expression of mast cell chymase by 50% compared with AngII infused mice lacking imatinib. Gene expression analysis of SMC marker SM22a demonstrated an increase by 48% together with a more intact medial layer after treatment with imatinib as evaluated with SM22 alpha immunostaining. Conclusion: Present findings highlight the importance of tyrosine kinase pathways in the development of AAA. Our results show, that imatinib treatment inhibits essential mast cell, T lymphocyte and SMC mediated processes in experimental AAA. Thus, our results support the idea that tyrosine kinase inhibitors may be useful in the treatment of pathological vascular inflammation and remodeling in conditions like AAA. (C) 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
  • Emameh, Reza Zolfaghari; Kuuslahti, Marianne; Näreaho, Anu; Sukura, Antti; Parkkila, Seppo (2016)
    Trichinellosis is a helminthic infection where different species of Trichinella nematodes are the causative agents. Several molecular assays have been designed to aid diagnostics of trichinellosis. These assays are mostly complex and expensive. The genomes of Trichinella species contain certain parasite-specific genes, which can be detected by polymerase chain reaction (PCR) methods. We selected -carbonic anhydrase (-CA) gene as a target, because it is present in many parasites genomes but absent in vertebrates. We developed a novel -CA gene-based method for detection of Trichinella larvae in biological samples. We first identified a -CA protein sequence from Trichinella spiralis by bioinformatic tools using -CAs from Caenorhabditis elegans and Drosophila melanogaster. Thereafter, 16 sets of designed primers were tested to detect -CA genomic sequences from three species of Trichinella, including T.spiralis, Trichinellapseudospiralis and Trichinellanativa. Among all 16 sets of designed primers, the primer set No. 2 efficiently amplified -CA genomic sequences from T.spiralis, T.pseudospiralis and T.nativa without any false-positive amplicons from other parasite samples including Toxoplasma gondii, Toxocara cati and Parascaris equorum. This robust and straightforward method could be useful for meat inspection in slaughterhouses, quality control by food authorities and medical laboratories.