Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.

• Publikační typ
• časopisecké články MeSH

The human Nek2 protein kinase is the closest known mammalian relative of the mitotic regulator NIMA of Aspergillus nidulans. The two kinases share 47% sequence identity over their catalytic domains and display a similar cell cycle-dependent expression peaking at the G2 to M phase transition. Hence, it is attractive to speculate that human Nek2 and fungal NIMA may carry out similar functions at the onset of mitosis. To study the biochemical properties and substrate specificity of human Nek2 and compare them to those reported previously for other NIMA-related protein kinases, we have expressed Nek2 in insect cells. We show that recombinant Nek2 is active as a serine/threonine-specific protein kinase and may undergo autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a similar, albeit not identical, set of proteins and synthetic peptides, and beta-casein was found to be a suitable substrate for assaying Nek2 in vitro. By exploiting these findings, we have studied the cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2 activity parallels its abundance, being low during M and G1 but high during S and G2 phase. Taken together, our results suggest that human Nek2 resembles fungal NIMA in its primary structure, cell cycle regulation of expression, and substrate specificity, but that Nek2 may function earlier in the cell cycle than NIMA.

• MeSH
• Aspergillus nidulans genetika   MeSH
• Baculoviridae genetika   MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• fosforylace MeSH
• HeLa buňky metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• peptidové fragmenty metabolismus   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny buněčného cyklu * MeSH
• rekombinantní proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• Spodoptera cytologie   MeSH
• substrátová specifita MeSH
• tyrosinkinasy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Oocyte ageing is the most important factor affecting egg quality of several fish species after ovulation. Oxidative stress has been proposed as the initiator of the oocyte ageing process in other vertebrates. To identify the role of oxidative stress and apoptosis on the progress of oocyte ageing in the common carp Cyprinus carpio, changes in the relative mRNA abundance of selected transcripts were examined. The possible alteration in the oxidation status of the oocytes during ageing was also studied. In addition, the activity of antioxidant enzymes during oocyte ageing was evaluated. Oocytes from 6 females were incubated in vivo for 14 hours post-ovulation (HPO) and in vitro for 10 hours post-stripping (HPS) at 20°C before fertilization. Hatching rates were over 65% up to 4-6 HPO, finally dropping to 1.3% at 12-14 HPO.Hatching rates were over 65% up to 4-6 HPO, finally dropping to 1.3% at 12-14 HPO. Hatching rates were more than 70% for the eggs stored in vitro up to 6 HPS and then decreased to 21.3% at 10 HPS. The results demonstrated no significant changes in the relative mRNA levels of oxidative stress-related genes or genes involved in the cell cycle during the progress of oocyte ageing in common carp. Additionally, the amount of TBARS and carbonyls did not change as time elapsed following ovulation. The apoptosis-related genes however, were significantly altered following the prolonged time interval between ovulation and fertilization. The lack of response of both activities of antioxidant enzymes and oxidation products during oocyte ageing strengthens the conclusion that oxidative stress is unlikely to be a main factor determining the progress of oocyte ageing in common carp. However, an increase in the mRNA abundance of apoptosis-related genes demonstrates that apoptotic pathway might be involved in the progress of oocyte ageing.

• MeSH
• antioxidancia metabolismus   MeSH
• kapři metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• oocyty cytologie   metabolismus   MeSH
• oxidace-redukce MeSH
• oxidoreduktasy metabolismus   MeSH
• rybí proteiny metabolismus   MeSH
• stárnutí buněk * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Events occurring in the chicken caecum following Salmonella Enteritidis infection are relatively well-described. However, mechanisms of the immune response and defence beyond the intestinal tract are less well-described. In this study, we therefore determined changes in protein abundance in the liver and blood serum in response to S. Enteritidis infection using the unbiased approach of shotgun proteomics. Complement and coagulation cascades, TNF signalling, antigen processing and presentation was activated in the liver following infection with S. Enteritidis. Chicken proteins that decreased in the liver were involved in glycolysis, the citrate cycle, oxidative phosphorylation and fatty acid metabolism. No functional category was significantly activated or suppressed in the serum. Concerning individual proteins, VNN1, SAA, AVD, SERPINA3, SERPINB10, AGT, MRP126 or CP increased in abundance both in the liver and serum. MT4, MT3, PTGDS, GLRX and TGM4, though highly inducible in the liver, did not increase in the serum. PIGR, SERPINF2 and IGJ increased in the serum but not in the liver. SERPINA4, apoAIV, CLEC3B, SERPINF1, HRG, AHSG and ALB decreased both in the liver and serum. Avidin-like LOC431660, THRSP, GATM, GGACT, ACOX1, ALDOB or FABP7 decreased in the liver but not in the serum. Finally, CKM, CKB, PLTP, COMP, IGFALS, AMY1A or SERPIND1 decreased in the serum after S. Enteritidis infection but not in the liver. Differently abundant proteins characterise the chicken's response to infection and can be also used as markers of chicken health status.

• MeSH
• cékum imunologie   MeSH
• játra imunologie   metabolismus   mikrobiologie   MeSH
• kur domácí krev   imunologie   MeSH
• nemoci drůbeže imunologie   mikrobiologie   MeSH
• prezentace antigenu MeSH
• proteiny akutní fáze analýza   MeSH
• proteomika * MeSH
• Salmonella enteritidis MeSH
• salmonelová infekce u zvířat krev   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cyclin D1 is a cell-cycle regulator essential for G1 phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumour types, including breast carcinomas. In spite of the accumulating genetic evidence, however, there are no data regarding abundance and properties of the cyclin D1 protein in breast cancer. We now report aberrant nuclear overexpression/accumulation of the cyclin D1 protein in about half of the 170 primary breast carcinoma specimens analyzed by monoclonal antibody immunohistochemistry, indicating that the frequency of cyclin D1 abnormalities may be considerably higher than previously deduced from DNA amplification studies. A comparison of the expression patterns in matched lesions at different stages of tumour progression revealed that the cyclin D1 protein aberration appears to reflect a relatively early event and that, when acquired by a tumour, it is maintained throughout breast cancer progression including metastatic spread. In both tumour tissues and breast cancer cell lines, the abundance of this protein shows characteristic variations consistent with a cell-cycle oscillation and the peak levels expressed in G1. In all 7 cell lines whose retinoblastoma (Rb) protein is mutant or complexed to SV40 T antigen, exceptionally low levels of cyclin D1 protein and mRNA were found. Antibody-mediated and anti-sense oligonucleotide knockout experiments demonstrate the requirement for the cell-cycle regulatory function of cyclin D1 in breast cancer lines with single or multiple copies of the gene and reveal the absence of such a requirement in the cell lines with Rb defects. Our data are consistent with the notion that the emerging "Rb-cyclin D1 pathway" represents a frequent target of oncogenic abnormalities in breast cancer.

• MeSH
• buněčný cyklus MeSH
• cyklin D1 MeSH
• cykliny analýza   fyziologie   MeSH
• geny retinoblastomu MeSH
• lidé MeSH
• messenger RNA analýza   MeSH
• molekulární sekvence - údaje MeSH
• nádory prsu * genetika   chemie   patologie   MeSH
• onkogenní proteiny analýza   fyziologie   MeSH
• prsy * chemie   patologie   MeSH
• RNA nádorová analýza   MeSH
• sekvence nukleotidů MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Wnt1 inducible protein-1 signaling pathway (WISP-1) is a relatively new adipokine involved in many cellular processes, including epithelial mucosa healing. The aim of the study was to compare circulating levels of WISP-1 and other selected adipokines [adiponectin, resistin and retinol-binding protein 4 (RBP-4)] in children with inflammatory bowel disease (IBD) with healthy controls and to investigate possible differences between Crohn's disease patients. (CD) or ulcerative colitis (UC). The study was performed as a case-control study. In addition to adipokines, anthropometric, lipid parameters, markers of inflammation or disease activity were evaluated in all participants. Compared to healthy controls (n=20), significantly lower levels of adiponectin and higher levels of resistin and WISP-1 were found in patients with IBD (n=58). Elevation of WISP-1 was detected only in the CD group (n=31). There were no differences in RBP-4 levels between the groups. Adiponectin, WISP-1 and RBP-4 were independently associated with body mass index only, resistin levels were associated with C-reactive protein levels and leukocyte counts. Adverse adipokines production reflects presence of dysfunctional fat tissue in IBD patients. Higher levels of WISP-1 in CD compared to patients with UC may indicate a specific role for mesenteric adipose tissue in WISP-1 production.

• MeSH
• adipokiny MeSH
• adiponektin MeSH
• dítě MeSH
• idiopatické střevní záněty * MeSH
• lidé MeSH
• mezibuněčné signální proteiny CCN krev   MeSH
• protoonkogenní proteiny krev   MeSH
• resistin MeSH
• signální transdukce MeSH
• studie případů a kontrol MeSH
• ulcerózní kolitida * MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Knowledge of conditions affecting sperm quality is essential for efficient culture of fish for commercial purposes and conservation of species. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry were used to characterize the proteomic profile of Acipenser dabryanus spermatozoa relative to motility and fertilization capacity. There were differential amounts of protein in 313 spots in spermatozoa of males classified to have relatively greater or lesser spermatozoa quality. The functions of 43 of 50 selected proteins were identified. The proteins in 14 spots were involved in metabolism, and of these, proteins in 11 spots were highly abundant in spermatozoa of males categorized to have spermatozoa of greater quality, including pyruvate kinase, enolase B, phosphoglycerate kinase, lactate dehydrogenase, cytosolic malate dehydrogenase, brain creatine kinase b, Ckmb protein, and nucleoside diphosphate kinase. The proteins involved in mechanics of flagellum movement were identified, including the dynein intermediate chain, radial spoke head 1 homolog; ropporin-1-like, Bardet-Biedl syndrome 5, ADP-ribosylation factor-like protein 3, tektin-4, gamma-actin, and tubulin cytoskeleton proteins to be differentially abundant in spermatozoa that were classified relatively greater or lesser quality. Heat shock proteins, copper/zinc superoxide dismutase and peroxiredoxins, which are involved in stress response were of differential abundance in spermatozoa from males with spermatozoa in the two different classification groups. Proteins were also detected that are involved in protein folding and binding, or hydrolase activity. The results are valuable for the prediction of sperm quality and for reproduction management in A. dabryanus and other threatened species.

• MeSH
• motilita spermií MeSH
• proteomika MeSH
• rybí proteiny chemie   metabolismus   MeSH
• ryby fyziologie   MeSH
• spermie chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17β-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2.

• MeSH
• apolipoprotein C-II genetika   MeSH
• chemické látky znečišťující vodu toxicita   MeSH
• cytochrom P-450 CYP3A genetika   MeSH
• estradiol toxicita   MeSH
• glutathiontransferasa genetika   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• hypolipidemika toxicita   MeSH
• kultivované buňky MeSH
• kyselina klofibrová toxicita   MeSH
• messenger RNA metabolismus   MeSH
• modulátory estrogenních receptorů toxicita   MeSH
• Oncorhynchus mykiss * MeSH
• rybí proteiny genetika   MeSH
• systém (enzymů) cytochromů P-450 genetika   MeSH
• vitelogeniny genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

• MeSH
• hnědá tuková tkáň cytologie   metabolismus   MeSH
• hnědé tukové buňky cytologie   metabolismus   MeSH
• lopatka cytologie   metabolismus   MeSH
• myši MeSH
• proteiny genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• separace buněk metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The review is focused on plant proteome response to salinity with respect to physiological aspects of plant salt stress response. The attention is paid to both osmotic and ionic effects of salinity stress on plants with respect to several protein functional groups. Therefore, the role of individual proteins involved in signalling, changes in gene expression, protein biosynthesis and degradation and the resulting changes in protein relative abundance in proteins involved in energy metabolism, redox metabolism, stress- and defence-related proteins, osmolyte metabolism, phytohormone, lipid and secondary metabolism, mechanical stress-related proteins as well as protein posttranslational modifications are discussed. Differences between salt-sensitive (glycophytes) and salt-tolerant (halophytes) plants are analysed with respect to differential salinity tolerance. In conclusion, contribution of proteomic studies to understanding plant salinity tolerance is summarised and discussed.

• Publikační typ
• časopisecké články MeSH