The characterization of the immune response of chickens to Salmonella infection is usually limited to the quantification of expression of genes coding for cytokines, chemokines or antimicrobial peptides. However, processes occurring in the cecum of infected chickens are likely to be much more diverse. In this study we have therefore characterized the transcriptome and proteome in the chicken cecum after infection with Salmonella Enteritidis. Using a combination of 454 pyrosequencing, protein mass spectrometry and quantitative real-time PCR, we identified 48 down- and 56 up-regulated chicken genes after Salmonella Enteritidis infection. The most inducible gene was that coding for MMP7, exhibiting a 5952 fold induction 9 days post-infection. An induction of greater than 100 fold was observed for IgG, IRG1, SAA, ExFABP, IL-22, TRAP6, MRP126, IFNγ, iNOS, ES1, IL-1β, LYG2, IFIT5, IL-17, AVD, AH221 and SERPIN B. Since prostaglandin D2 synthase was upregulated and degrading hydroxyprostaglandin dehydrogenase was downregulated after the infection, prostaglandin must accumulate in the cecum of chickens infected with Salmonella Enteritidis. Finally, above mentioned signaling was dependent on the presence of a SPI1-encoded type III secretion system in Salmonella Enteritidis. The inflammation lasted for 2 weeks after which time the expression of the "inflammatory" genes returned back to basal levels and, instead, the expression of IgA and IgG increased. This points to an important role for immunoglobulins in the restoration of homeostasis in the cecum after infection.

• MeSH
• cékum imunologie   metabolismus   MeSH
• hmotnostní spektrometrie veterinární   MeSH
• kur domácí * MeSH
• nemoci drůbeže genetika   imunologie   mikrobiologie   MeSH
• nemoci úst genetika   imunologie   mikrobiologie   veterinární   MeSH
• northern blotting veterinární   MeSH
• polymerázová řetězová reakce veterinární   MeSH
• přirozená imunita * MeSH
• proteom imunologie   MeSH
• ptačí proteiny genetika   imunologie   MeSH
• regulace genové exprese * MeSH
• Salmonella enteritidis imunologie   MeSH
• salmonelová infekce u zvířat genetika   imunologie   mikrobiologie   MeSH
• sekvenční analýza DNA veterinární   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH
• ptačí proteiny MeSH

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

• Klíčová slova
• Grow yeast cultures, Polysome profile analysis, Sucrose density gradient centrifugation, Western and Northern blotting, Yeast whole cell extracts (WCEs),
• MeSH
• centrifugace - gradient hustoty metody   MeSH
• northern blotting metody   MeSH
• polyribozomy chemie   genetika   MeSH
• Saccharomyces cerevisiae chemie   cytologie   genetika   MeSH
• sacharosa chemie   MeSH
• western blotting metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sacharosa MeSH

An extraordinary variation in mitochondrial DNA sequence exists in angiosperm Silene vulgaris. The atp1 gene is flanked by very variable regions, as deduced from four completely sequenced mitochondrial genomes of this species. This diversity contributed to a highly variable transcript profile of this gene observed across S. vulgaris populations. We examined the atp1 transcript in the KOV mitochondrial genome and found three 5' ends, created most likely by the combination of transcription initiation and RNA processing. Most atp1 transcripts terminated about 70 bp upstream of the translation stop codon, which was present in only 10 % of them. Controlled crosses between a KOV mother and a geographically distant pollen donor (Krasnoyarsk, Russia) showed that nuclear background also affected atp1 transcription. The distant pollen donor introduced the factor(s) preventing the formation of a long 2,100 nt-transcript, because this long atp1 transcript reappeared in the progeny from self-crosses. The highly rearranged mitochondrial genomes with a variation in gene flanking regions make S. vulgaris an excellent model for the study of mitochondrial gene expression in plants.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• buněčné jádro genetika   MeSH
• genetická transkripce * MeSH
• genom mitochondriální genetika   MeSH
• genom rostlinný genetika   MeSH
• genová přestavba genetika   MeSH
• křížení genetické MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• northern blotting MeSH
• otevřené čtecí rámce genetika   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Silene genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• messenger RNA MeSH
• rostlinné proteiny MeSH

Hypertension in humans and experimental models has a strong hereditary basis, but identification of causative genes remains challenging. Quantitative trait loci (QTLs) for hypertension and salt sensitivity have been reported on rat chromosome 18. We set out to genetically isolate and prioritize genes within the salt-sensitivity and hypertension QTLs on the spontaneously hypertensive rat (SHR) chromosome 18 by developing and characterizing a series of congenic strains derived from the SHR and normotensive Brown Norway rat strains. The SHR.BN-D18Rat113/D18Rat82 congenic strain exhibits significantly lower blood pressure and is salt resistant compared with the SHR. Transplantation of kidneys from SHR.BN-D18Rat113/D18Rat82 donors into SHR recipients is sufficient to attenuate increased blood pressure but not salt sensitivity. Derivation of congenic sublines allowed for the separation of salt sensitivity from hypertension QTL regions. Renal expression studies with microarray and Solexa-based sequencing in parental and congenic strains identified 4 differentially expressed genes within the hypertension QTL region, one of which is an unannotated transcript encoding a previously undescribed, small, nonprotein coding RNA. Sequencing selected biological candidate genes within the minimal congenic interval revealed a nonsynonymous variant in SHR transcription factor 4. The minimal congenic interval is syntenic to a region of human chromosome 18 where significant linkage to hypertension was observed in family based linkage studies. These congenic lines provide reagents for identifying causative genes that underlie the chromosome 18 SHR QTLs for hypertension and salt sensitivity. Candidate genes identified in these studies merit further investigation as potentially causative hypertension genes in SHR and human hypertension.

• MeSH
• genetická predispozice k nemoci genetika   MeSH
• hypertenze etiologie   genetika   patofyziologie   MeSH
• jednonukleotidový polymorfismus MeSH
• krevní tlak genetika   fyziologie   MeSH
• krysa rodu Rattus MeSH
• kuchyňská sůl škodlivé účinky   MeSH
• ledviny metabolismus   MeSH
• lokus kvantitativního znaku genetika   MeSH
• mapování chromozomů MeSH
• northern blotting MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani inbrední BN MeSH
• potkani inbrední SHR MeSH
• proteiny regulující apoptózu genetika   MeSH
• receptor melanokortinový typ 2 genetika   MeSH
• receptor melanokortinový typ 4 genetika   MeSH
• receptory melanokortinové genetika   MeSH
• savčí chromozomy genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• transplantace ledvin metody   MeSH
• tyrosinfosfatasa nereceptorového typu 2 genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• CIDEA protein, rat MeSH Prohlížeč
• kuchyňská sůl MeSH
• melanocortin 5 receptor MeSH Prohlížeč
• proteiny regulující apoptózu MeSH
• Ptpn2 protein, rat MeSH Prohlížeč
• receptor melanokortinový typ 2 MeSH
• receptor melanokortinový typ 4 MeSH
• receptory melanokortinové MeSH
• tyrosinfosfatasa nereceptorového typu 2 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

Maturation of Norway spruce (Picea abies L.) somatic embryos is induced by abscisic acid (ABA). Several proteins were proven to be involved in ABA sensing including ABI3/VP1 transcription factors and their orthologue PaVP1 was characterized in spruce. To evaluate the role of PaVP1 both in embryogenic potential and in the process of embryo maturation, we studied PaVP1 expression in lines with contrast embryogenic capacities in parallel with detailed anatomical characterization. PaVP1 expression was determined by northern blot hybridisation, which revealed presence of two differentially regulated VP1-like B3-domain transcripts. Full-length PaVP1 transcript level was negligible in all lines on the proliferation media, but it differed strongly on the maturation media containing ABA. In non-embryogenic line, lacking any differentiated structures, the transcript remained undetectable. In contrast, in embryogenic lines with meristematic centres attached to suspensor cells, PaVP1 expression increased strongly after transition onto the maturation media. In highly embryogenic lines, it kept on a high level until the embryos reached cotyledonary stage, while in developmentally arrested line incapable to form mature embryos, the expression dropped down in connection with advanced disintegration of the meristematic centres. Removal of ABA from the maturation media after 2 weeks of maturation resulted in aberrant embryo development and rapid decrease in PaVP1 expression, indicating the impact of exogenously supplemented ABA on both initiation and maintenance of PaVP1 expression and proper embryo development. Since permanently high or increasing PaVP1 transcript levels accompanied proper embryo development in all experiments, it could be regarded as a good marker of this process.

• MeSH
• alternativní sestřih MeSH
• genetická transkripce účinky léků   MeSH
• kyselina abscisová farmakologie   MeSH
• northern blotting MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• rostlinné proteiny genetika   fyziologie   MeSH
• smrk účinky léků   embryologie   genetika   MeSH
• transkripční faktory genetika   fyziologie   MeSH
• vývojová regulace genové exprese účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina abscisová MeSH
• rostlinné proteiny MeSH
• transkripční faktory MeSH

tmRNA and protein SmpB are the main components required for rescue of stalled ribosomes incapable of properly elongating or terminating the polypeptide chain. We examined the tmRNA level and protein synthesis in Streptomyces aureofaciens, S. griseus and S. collinus synthesizing tetracycline, streptomycin and kirromycin, respectively, during various stress conditions. Downshift in temperature caused a decrease in protein synthesis but the level of tmRNA increased. Shift up in temperature induced decay of tmRNA in all strains and in S. collinus led to stimulation and in S. aureofaciens and S. griseus to inhibition of protein synthesis. At high NaCl concentrations protein synthesis was inhibited and tmRNA decayed. Shift in pH from 7.0 to 5.0 had no pronounced effect on the tmRNA level while upshift to pH 9.0 in S. collinus and S. aureofaciens caused inhibition of protein synthesis and decay of tmRNA in S. collinus. In contrast, protein synthesis and tmRNA level increased in S. griseus at the alkaline pH. Our data show that tmRNA abundance is important for survival of streptomycetes under certain unfavorable conditions.

• MeSH
• bakteriální RNA metabolismus   MeSH
• chlorid sodný škodlivé účinky   MeSH
• koncentrace vodíkových iontů MeSH
• messenger RNA metabolismus   MeSH
• northern blotting MeSH
• proteosyntéza MeSH
• RNA transferová metabolismus   MeSH
• Streptomyces aureofaciens fyziologie   MeSH
• Streptomyces fyziologie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• chlorid sodný MeSH
• messenger RNA MeSH
• RNA transferová MeSH
• tmRNA MeSH Prohlížeč

Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNA(i)(Met)), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5' untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo "snapshot" of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.

• MeSH
• buněčné extrakty MeSH
• eukaryotické iniciační faktory chemie   MeSH
• formaldehyd chemie   MeSH
• frakcionace buněk metody   MeSH
• heparin chemie   MeSH
• iniciace translace peptidového řetězce fyziologie   MeSH
• northern blotting MeSH
• polyribozomy fyziologie   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• buněčné extrakty MeSH
• eukaryotické iniciační faktory MeSH
• formaldehyd MeSH
• heparin MeSH
• reagencia zkříženě vázaná MeSH

Decreased susceptibility of K. lactis mutants impaired in the function of cytochrome c, cytochrome c1 and cytochrome-c oxidase to fluconazole, bifonazole and amphotericin B in comparison with the isogenic wild-type strain was observed. Flow cytometry with rhodamine 6G did not show any changes in the accumulation of the dye in the mutant cells compared with the corresponding wild-type strain. Sterol analysis showed similar overall amount of sterols in both wild-type and mutant cells. Taking into account the increased amphotericin B resistance and significantly diminished susceptibility of mutant cells to lyticase digestion, the cell wall structure and/or composition may probably be responsible for the observed changes in the susceptibility of mutants to the antifungal compounds used.

• MeSH
• amfotericin B farmakologie   MeSH
• antifungální látky farmakologie   MeSH
• buněčná stěna chemie   účinky léků   MeSH
• endo-1,3-beta-glukanasa metabolismus   MeSH
• fungální léková rezistence * MeSH
• fungální proteiny genetika   metabolismus   MeSH
• Kluyveromyces cytologie   účinky léků   genetika   fyziologie   MeSH
• mikrobiální testy citlivosti MeSH
• mitochondrie účinky léků   genetika   fyziologie   MeSH
• multienzymové komplexy metabolismus   MeSH
• mutace * MeSH
• northern blotting MeSH
• proteasy metabolismus   MeSH
• průtoková cytometrie MeSH
• steroly metabolismus   MeSH
• transport elektronů účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfotericin B MeSH
• antifungální látky MeSH
• endo-1,3-beta-glukanasa MeSH
• fungální proteiny MeSH
• lyticase MeSH Prohlížeč
• multienzymové komplexy MeSH
• proteasy MeSH
• steroly MeSH

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

• MeSH
• biolistika metody   MeSH
• endonukleasy genetika   metabolismus   MeSH
• genotyp MeSH
• klonování DNA MeSH
• komplementární DNA chemie   genetika   MeSH
• konformace nukleové kyseliny MeSH
• listy rostlin genetika   metabolismus   virologie   MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin genetika   virologie   MeSH
• northern blotting MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• rostlinné viry genetika   patogenita   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• Solanum lycopersicum genetika   metabolismus   virologie   MeSH
• Solanum tuberosum genetika   metabolismus   virologie   MeSH
• viroidy genetika   patogenita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endonukleasy MeSH
• komplementární DNA MeSH
• malá interferující RNA MeSH
• RNA virová MeSH
• rostlinné proteiny MeSH