Non-healing wounds are a serious complication in diabetic patients. One of the detrimental factors contributing to limited wound healing is the accumulation of metalloproteinase-9 (MMP-9) in the wound. Selective inhibition of MMP-9 is one of the established therapeutic targets for diabetic wound healing. Here, a functional and biocompatible wound dressing is developed to enable a controlled release of a traceable vector loaded with the antisense siRNA against MMP-9 in the wound. The dressing consists of degradable polymer nanofibers embedded with a vector nanosystem - polymer-coated fluorescent nanodiamonds optimized for the binding of siRNA and colloidal stability of nanodiamond-siRNA complexes in a physiological environment. The developed dressing is tested on murine fibroblasts and also applied to wounds in a diabetic murine model to evaluate its suitability in terms of in vivo toxicity, biological efficacy, and handling. The treatment results in significant local inhibition of MMP-9 and a shortening of the wound healing time. The scar formation in treated diabetic-like mice becomes comparable with that in non-treated diabetes-free mice. Our results suggest that the application of our biocompatible dressing loaded with a non-toxic vector nanosystem is an effective and promising approach to gene therapy of non-healing wounds.

• MeSH
• aplikace lokální MeSH
• experimentální diabetes mellitus * MeSH
• hojení ran * účinky léků   MeSH
• malá interferující RNA * aplikace a dávkování   genetika   farmakologie   MeSH
• matrixová metaloproteinasa 9 metabolismus   genetika   MeSH
• myši MeSH
• obvazy MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malá interferující RNA * MeSH
• matrixová metaloproteinasa 9 MeSH

Maintenance of genome stability is essential for every living cell as genetic information is repeatedly challenged during DNA replication in each cell division event. Errors, defects, delays, and mistakes that arise during mitosis or meiosis lead to an activation of DNA repair processes and in case of their failure, programmed cell death, i.e. apoptosis, could be initiated. Fam208a is a protein whose importance in heterochromatin maintenance has been described recently. In this work, we describe the crucial role of Fam208a in sustaining the genome stability during the cellular division. The targeted depletion of Fam208a in mice using CRISPR/Cas9 leads to embryonic lethality before E12.5. We also used the siRNA approach to downregulate Fam208a in zygotes to avoid the influence of maternal RNA in the early stages of development. This early downregulation increased arresting of the embryonal development at the two-cell stage and occurrence of multipolar spindles formation. To investigate this further, we used the yeast two-hybrid (Y2H) system and identified new putative interaction partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp. Their co-expression with Fam208a was assessed by qRT-PCR profiling and in situ hybridisation [1] in multiple murine tissues. Based on these results we proposed that Fam208a functions within the HUSH complex by interaction with Mphosph8 as these proteins are not only able to physically interact but also co-localise. We are bringing new evidence that Fam208a is multi-interacting protein affecting genome stability on the level of cell division at the earliest stages of development and also by interaction with methylation complex in adult tissues. In addition to its epigenetic functions, Fam208a appears to have an additional role in zygotic division, possibly via interaction with newly identified putative partners Gpsm2, Amn1, Eml1, Svil, and Itgb3bp.

• Klíčová slova
• Fam208a, Genome stability, HUSH, Multipolar spindle apparatus,
• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• buněčné dělení genetika   fyziologie   MeSH
• CRISPR-Cas systémy MeSH
• embryonální vývoj genetika   fyziologie   MeSH
• fosfoproteiny metabolismus   MeSH
• HEK293 buňky MeSH
• jaderné proteiny fyziologie   MeSH
• letální geny MeSH
• lidé MeSH
• malá interferující RNA genetika   farmakologie   MeSH
• multiproteinové komplexy MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nestabilita genomu MeSH
• RNA interference MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• zygota metabolismus   MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• odvolaná publikace MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Fam208a protein, mouse MeSH Prohlížeč
• fosfoproteiny MeSH
• jaderné proteiny MeSH
• malá interferující RNA MeSH
• MPHOSPH8 protein, human MeSH Prohlížeč
• Mphosph8 protein, mouse MeSH Prohlížeč
• multiproteinové komplexy MeSH
• TASOR protein, human MeSH Prohlížeč

Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.

• Klíčová slova
• chemotaxis, membrane rafts,
• MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buňky NK cytologie   metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• chemotaxe leukocytů fyziologie   MeSH
• detergenty farmakologie   MeSH
• kultivované buňky MeSH
• lektinové receptory NK-buněk - podrodina C metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina K fyziologie   MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• membránové mikrodomény účinky léků   fyziologie   MeSH
• mikrofilamenta fyziologie   MeSH
• mikrofilamentové proteiny antagonisté a inhibitory   genetika   fyziologie   MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické metabolismus   MeSH
• RNA interference MeSH
• signální transdukce imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• detergenty MeSH
• HCST protein, human MeSH Prohlížeč
• KLRK1 protein, human MeSH Prohlížeč
• LCP1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina C MeSH
• lektinové receptory NK-buněk - podrodina K MeSH
• malá interferující RNA MeSH
• mikrofilamentové proteiny MeSH
• multiproteinové komplexy MeSH
• proteom MeSH
• receptory imunologické MeSH

OBJECTIVE: The regulator of G-protein signaling (RGS) molecules represent a class of proteins that modulate the signaling activity of G-protein coupled receptors. Regulator of G-protein signaling 4 (RGS4) is of particular interest in schizophrenia due to reported downregulation of RGS4 transcripts in schizophrenia as well as a connection between RGS4 and a number of receptors implicated in schizophrenia. The mechanism of RGS4 involvement in the pathophysiology of this illness is not clear. METHODS: To elucidate thise role of RGS4 in pathophysiology of schizophrenia, we silenced RGS4 using siRNAs in human neuroblastoma cell lines and we studied the effects of differential RGS4 expression by microarray. RESULTS AND CONCLUSION: The cell lines with downregulated expression of RGS4 showed 67 genes with changed expression (30 underexpressed and 37 overexpressed). We have detected three subgroups of genes which might be implicated in schizophrenia pathophysiology: histone genes, which suggest epigenetic mechanisms of the disease; genes for transcription factors associated with other genes relevant to schizophrenia pathology (BDNF and DISCI1) and a heterogeneous group containing genes for G-proteins (GPR50 and GPR64) and calcium binding proteins.

• MeSH
• buněčné linie MeSH
• down regulace MeSH
• genetická transkripce MeSH
• genom lidský genetika   MeSH
• histony genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• mikročipová analýza MeSH
• mozkový neurotrofický faktor genetika   MeSH
• neurony účinky léků   metabolismus   MeSH
• proteiny nervové tkáně genetika   MeSH
• proteiny RGS genetika   MeSH
• receptory spřažené s G-proteiny genetika   MeSH
• schizofrenie genetika   MeSH
• stanovení celkové genové exprese MeSH
• umlčování genů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADGRG2 protein, human MeSH Prohlížeč
• GPR50 protein, human MeSH Prohlížeč
• histony MeSH
• malá interferující RNA MeSH
• mozkový neurotrofický faktor MeSH
• proteiny nervové tkáně MeSH
• proteiny RGS MeSH
• receptory spřažené s G-proteiny MeSH
• RGS4 protein MeSH Prohlížeč

Translocation (12;21), the most frequent chromosomal aberration in childhood acute lymphoblastic leukemia, creates TEL/AML1 fusion gene. Resulting hybrid protein was shown to have a role in pre-leukemia establishment. To address its role for leukemic cell survival, we applied RNA interference to silence TEL/AML1 in leukemic cells. We designed and tested 11 different oligonucleotides targeting the TEL/AML1 fusion site. Using most efficient siRNAs, we achieved an average of 74-86% TEL/AML1 protein knockdown in REH and UOC-B6 leukemic cells, respectively. TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, it resulted in the modest but significant increase in the S phase fraction and in higher proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced by AML1 silencing, thus, supporting our hypothesis that TEL/AML1 may block AML1-mediated promotion of G1/S progression through the cell cycle. In line with the lack of major effect on phenotype, we found no significant changes in clonogenic potential and global gene expression pattern upon TEL/AML1 depletion. Our data suggest that though TEL/AML1 is important for the (pre)leukemic clone development, it may be dispensable for leukemic cell survival and would not be a suitable target for gene-specific therapy.

• MeSH
• buněčné klony MeSH
• buněčný cyklus MeSH
• fúzní onkogenní proteiny genetika   fyziologie   MeSH
• leukemie patologie   MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• nádorové buněčné linie MeSH
• protein PEBP2A2 genetika   fyziologie   MeSH
• RNA interference * MeSH
• viabilita buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fúzní onkogenní proteiny MeSH
• malá interferující RNA MeSH
• protein PEBP2A2 MeSH
• TEL-AML1 fusion protein MeSH Prohlížeč

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H(2), is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.

• MeSH
• biologický transport MeSH
• cyklooxygenasa 1 genetika   MeSH
• intramolekulární oxidoreduktasy genetika   MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• messenger RNA analýza   MeSH
• nádorové buněčné linie MeSH
• nádory děložního čípku enzymologie   patologie   MeSH
• prostaglandiny metabolismus   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům genetika   MeSH
• regulace genové exprese enzymů * MeSH
• syntázy prostaglandinu E MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABCC4 protein, human MeSH Prohlížeč
• cyklooxygenasa 1 MeSH
• intramolekulární oxidoreduktasy MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH
• prostaglandiny MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům MeSH
• PTGES protein, human MeSH Prohlížeč
• syntázy prostaglandinu E MeSH

Due to a complete lack of the tRNA genes in the mitochondrial genome of Trypanosoma brucei, all tRNAs needed for mitochondrial translation have to be imported into the organelle from the cytosol. A previous study showed that the modified nucleotide s(2)U could act as a negative determinant for mitochondrial tRNA import in another kinetoplastid, Leishmania tarentolae. We have investigated whether the same type of cytosolic control for tRNA retention exists in T. brucei. Based on Northern analysis with subcellular RNA fractions and in vitro import assays, we demonstrate that silencing of the cysteine desulfurase, TbNfs (TbIscS), the key enzyme in tRNA thiolation (s(2)U) and Fe-S cluster formation in vivo, has no effect on tRNA partitioning. This observation is especially surprising in light of a recent report suggesting that in L. tropica the Rieske Fe-S protein is an essential component of the RNA import complex (RIC). In line with the above observation, we also show that down-regulation of the Rieske protein by RNA interference, similar to the TbNfs knockdowns, has no effect on import. The data presented here supports the view that in T. brucei: (1) s(2)U is not a negative determinant for tRNA import; (2) the Rieske protein is not an essential component of the import machinery, and (3) since the Rieske protein is essential for respiration and maintenance of inner mitochondrial membrane potential, neither process plays a critical role in tRNA import. We therefore suggest that the T. brucei import machinery differs substantially from what has been described in Leishmania.

• MeSH
• cytosol metabolismus   MeSH
• imunoblotting MeSH
• lyasy štěpící vazby C-S antagonisté a inhibitory   genetika   metabolismus   MeSH
• malá interferující RNA farmakologie   MeSH
• mitochondrie genetika   metabolismus   MeSH
• northern blotting MeSH
• respirační komplex III metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• RNA transferová genetika   metabolismus   MeSH
• síra metabolismus   MeSH
• subcelulární frakce MeSH
• Trypanosoma brucei brucei genetika   růst a vývoj   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• cysteine desulfurase MeSH Prohlížeč
• lyasy štěpící vazby C-S MeSH
• malá interferující RNA MeSH
• respirační komplex III MeSH
• Rieske iron-sulfur protein MeSH Prohlížeč
• RNA protozoální MeSH
• RNA transferová MeSH
• síra MeSH

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• down regulace MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• mapování interakce mezi proteiny MeSH
• nádorové buněčné linie MeSH
• protein TRAIL metabolismus   MeSH
• proteiny aktivující GTPasu fyziologie   MeSH
• receptory TNF metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• TRAIL receptory MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ARAP1 protein, human MeSH Prohlížeč
• malá interferující RNA MeSH
• protein TRAIL MeSH
• proteiny aktivující GTPasu MeSH
• receptory TNF MeSH
• TNFRSF10A protein, human MeSH Prohlížeč
• TNFSF10 protein, human MeSH Prohlížeč
• TRAIL receptory MeSH
• transportní proteiny MeSH

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F(1)F(o)-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc(1) complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F(1)F(o)-ATP synthase and NADH:ubiquinone oxidoreductase.

• MeSH
• 2D gelová elektroforéza MeSH
• adenosintrifosfát metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• hydrolýza MeSH
• imunoblotting MeSH
• imunoglobulin G imunologie   MeSH
• imunoprecipitace MeSH
• jaderné proteiny antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• kultivované buňky MeSH
• kur domácí MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• mitochondriální proteiny antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• mitochondriální protonové ATPasy antagonisté a inhibitory   biosyntéza   MeSH
• mitochondrie metabolismus   MeSH
• respirační komplex I antagonisté a inhibitory   biosyntéza   MeSH
• respirační komplex III metabolismus   MeSH
• respirační komplex IV antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• subcelulární frakce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• F1F0-ATP synthase MeSH Prohlížeč
• imunoglobulin G MeSH
• jaderné proteiny MeSH
• malá interferující RNA MeSH
• mitochondriální proteiny MeSH
• mitochondriální protonové ATPasy MeSH
• OXA1 protein MeSH Prohlížeč
• respirační komplex I MeSH
• respirační komplex III MeSH
• respirační komplex IV MeSH

Imatinib metylase is the first choice treatment for BCR/ABL positive chronic myelogenous leukemia (CML). However, as some CML patients develop resistance to imatinib therapy, there is a significant interest in development of alternative treatment strategies, such as identifying targets other than BCR/ABL that may participate in CML. Previously, we demonstrated strong PCNA up-regulation in CML patients. To further study its role in CML pathogenesis, we performed silencing of PCNA expression followed by array experiments. PCNA inhibition led to down-regulation of CDK1, CDK4, PLK1, ERK3, JNK1, STAT5, and several inhibitors of apoptosis (DAXX, Mdm2, survivin). The following genes were up-regulated: CDK inhibitors p21 and p19-INK4D, pro-apoptotic FAST kinase, fibronectin, etc. However, as PCNA affects cell growth in naturally proliferating cells as well as in cancerous cells, it seems to act a secondary role relating to proliferation activity of leukemic cells.

• MeSH
• apoptóza MeSH
• bcr-abl fúzní proteiny metabolismus   MeSH
• benzamidy MeSH
• chemorezistence MeSH
• chronická myeloidní leukemie farmakoterapie   genetika   MeSH
• imatinib mesylát MeSH
• lidé MeSH
• malá interferující RNA farmakologie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové biomarkery genetika   MeSH
• nádorové buňky kultivované MeSH
• piperaziny terapeutické užití   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proliferace buněk MeSH
• proliferační antigen buněčného jádra genetika   MeSH
• pyrimidiny terapeutické užití   MeSH
• regulace genové exprese u leukemie * MeSH
• RNA nádorová genetika   metabolismus   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * MeSH
• stanovení celkové genové exprese * MeSH
• umlčování genů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• benzamidy MeSH
• imatinib mesylát MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH
• nádorové biomarkery MeSH
• piperaziny MeSH
• proliferační antigen buněčného jádra MeSH
• pyrimidiny MeSH
• RNA nádorová MeSH