• MeSH
• arginin farmakologie   MeSH
• bilirubin farmakologie   MeSH
• hem * farmakologie   MeSH
• HIV-1 * fyziologie   účinky léků   MeSH
• latence viru fyziologie   MeSH
• lidé MeSH
• oxid uhelnatý MeSH
• RNA virová genetika   metabolismus   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

• MeSH
• játra chemie   patofyziologie   MeSH
• kadheriny chemie   MeSH
• myši MeSH
• posttranslační úpravy proteinů MeSH
• přetížení železem patofyziologie   MeSH
• retardační test MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.

• MeSH
• aktivinové receptory typu II metabolismus   MeSH
• deficit železa MeSH
• down regulace MeSH
• inhibitor diferenciace 1 genetika   metabolismus   MeSH
• injekce intraperitoneální MeSH
• játra účinky léků   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory morfogenetických kostních proteinů typu I metabolismus   MeSH
• receptory transferinu metabolismus   MeSH
• serinové endopeptidasy nedostatek   genetika   MeSH
• železo-dextranový komplex aplikace a dávkování   MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency. METHODS: C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv-/- mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit. RESULTS: Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv-/- mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA. DISCUSSION: Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.

• MeSH
• deficit železa MeSH
• dietní železo metabolismus   MeSH
• erythropoetin farmakologie   MeSH
• játra účinky léků   metabolismus   MeSH
• kostní morfogenetické proteiny genetika   metabolismus   MeSH
• krysa rodu rattus MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• přetížení železem genetika   metabolismus   MeSH
• signální transdukce účinky léků   genetika   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass. RESULTS: We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant. CONCLUSIONS: Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.

• MeSH
• extracelulární prostor metabolismus   MeSH
• genový knockout MeSH
• glykopeptidasa metabolismus   MeSH
• glykosylace MeSH
• játra cytologie   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   izolace a purifikace   metabolismus   MeSH
• myši MeSH
• neuraminidasa metabolismus   MeSH
• orgánová specificita MeSH
• svaly cytologie   metabolismus   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations of the TMPRSS6 gene, encoding the serine protease matriptase-2, lead to iron-refractory iron deficiency anemia. Matriptase-2 is a potent negative regulator of hepcidin. Based on in vitro data, it has recently been proposed that matriptase-2 decreases hepcidin synthesis by cleaving membrane hemojuvelin, a key protein of the hepcidin-regulatory pathway. However, in vivo evidence for this mechanism of action of matriptase-2 is lacking. To investigate the hemojuvelin-matriptase-2 interaction in vivo, an immunoblot assay for liver membrane hemojuvelin was optimized using hemojuvelin-mutant mice as a negative control. In wild-type mice, two hemojuvelin-specific bands of 35kDa and 20kDa were detected in mouse liver membrane fraction under reducing conditions; under non-reducing conditions, a single band of approximately 50kDa was seen. Phosphatidylinositol-specific phospholipase C treatment confirmed binding of the detected protein to the cell membrane by a glycosylphosphatidylinositol anchor, indicating that the major form of mouse liver membrane hemojuvelin is a glycosylphosphatidylinositol-bound heterodimer. Unexpectedly, comparison of liver homogenates from Tmprss6+/+ and Tmprss6-/- mice revealed significantly decreased, rather than increased, hemojuvelin heterodimer content in Tmprss6-/- mice. These data do not provide direct support for the concept that matriptase-2 cleaves membrane hemojuvelin and may indicate that, in vivo, the role of matriptase-2 in the regulation of hepcidin gene expression is more complex.

• MeSH
• anemie z nedostatku železa genetika   metabolismus   MeSH
• buněčná membrána genetika   metabolismus   MeSH
• dimerizace MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fosfolipasa C fosfoinositidové signalizace metabolismus   MeSH
• glykosylfosfatidylinositoly chemie   metabolismus   MeSH
• játra metabolismus   patologie   MeSH
• kationické antimikrobiální peptidy genetika   metabolismus   MeSH
• membránové proteiny nedostatek   genetika   metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• regulace genové exprese MeSH
• serinové endopeptidasy nedostatek   genetika   MeSH
• signální transdukce genetika   MeSH
• tkáňové extrakty chemie   MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

• MeSH
• časové faktory MeSH
• deficit železa MeSH
• dietní železo aplikace a dávkování   MeSH
• down regulace MeSH
• erytropoéza MeSH
• financování organizované MeSH
• flebotomie MeSH
• furin metabolismus   MeSH
• hypoxie buňky MeSH
• játra metabolismus   MeSH
• kationické antimikrobiální peptidy genetika   metabolismus   MeSH
• kosterní svaly metabolismus   MeSH
• krvácení etiologie   genetika   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   nedostatek   MeSH
• messenger RNA metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši knockoutované MeSH
• myši MeSH
• přetížení železem metabolismus   prevence a kontrola   MeSH
• serinové endopeptidasy metabolismus   MeSH
• železo MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

Growth inhibition of the DNA virus vaccinia (VACV) by NO is known to occur at the level of DNA synthesis. This inhibition is partially reversed by addition of deoxyribonucleosides, suggesting that NO or NO-related species inhibit viral ribonucleotide reductase (RR). However, the effect of NO on VACV-encoded RR or other DNA-synthesizing enzymes has not been demonstrated. In order to study the effects of NO on VACV-encoded RR, DNA polymerase (DNA pol) and thymidine kinase (TK), we generated a VACV recombinant expressing murine macrophage iNOS under control of a VACV early/late promoter p7.5. Using this recombinant, we demonstrate that expression of iNOS and the resulting production of NO inhibit activity of the viral RR, but not of viral DNA pol and TK. This NO-mediated inhibition of viral RR occurred around the same time as the increase of ADP levels, while it preceded the block in VACV DNA synthesis and the decrease of ATP levels. In addition, we tested the effects of DPTA/NONOate on the growth of different VACV mutants. Fold-inhibition of the growth of VACV deletion mutant for TK was comparable to that of wild-type VACV. VACV containing amplification of the gene for the small subunit of RR appeared to be least sensitive to DPTA/NONOate, while VACV deletion mutant for the large subunit of RR was most sensitive. The results provide a direct evidence for NO-mediated inhibition of VACV-encoded RR.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• alkeny metabolismus   MeSH
• buněčné linie MeSH
• Cercopithecus aethiops MeSH
• DNA virů metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• financování organizované MeSH
• mutace MeSH
• myši MeSH
• oxid dusnatý metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• ribonukleotidreduktasy antagonisté a inhibitory   metabolismus   MeSH
• synthasa oxidu dusnatého, typ II genetika   metabolismus   MeSH
• thymidinkináza metabolismus   MeSH
• virové proteiny antagonisté a inhibitory   metabolismus   MeSH
• virus vakcinie enzymologie   metabolismus   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH