Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

• MeSH
• cytokiny metabolismus   MeSH
• exprese genu MeSH
• Francisella tularensis * genetika   metabolismus   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   metabolismus   MeSH
• proteomika MeSH
• virulence genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.

• MeSH
• Bacteria enzymologie   patogenita   MeSH
• bakteriální infekce mikrobiologie   prevence a kontrola   MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální vakcíny imunologie   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy imunologie   metabolismus   MeSH
• lidé MeSH
• proteiny metabolismus   MeSH
• vazba proteinů MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

• MeSH
• delece genu * MeSH
• faktory virulence analýza   MeSH
• Francisella tularensis enzymologie   imunologie   patogenita   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy nedostatek   metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• mikrobiální viabilita MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• proteom analýza   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• vazba proteinů MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.

• MeSH
• dospělí MeSH
• fluorescenční mikroskopie MeSH
• fluorescenční protilátková technika nepřímá MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   MeSH
• imunohistochemie MeSH
• keratiny biosyntéza   genetika   metabolismus   MeSH
• konjunktiva metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• limbus corneae metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• RNA biosyntéza   genetika   MeSH
• rohovka metabolismus   MeSH
• rohovkový epitel metabolismus   MeSH
• senioři MeSH
• skléra metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cancer cell lines derived from hepatocytes have an altered phenotype and they lack hepatocyte-specific functions. It is at least partly due to the under-expression of transcription factors such as hepatocyte nuclear factor 4α (HNF4α), steroid receptor co-activator 1 (SRC1) etc. Recently, a strategy of transient transfection of human hepatic cells with HNF4α revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes. In the current study we established a human cell line derived from HepG2 cells stably transfected with human HNF4α, and we examined this line for hepatospecific markers. Of the 9 clones analyzed, we found an increased secretion of fibrinogen (9 clones), albumin (5 clones) and plasminogen (3 clones), while secretion of alpha1-antitrypsin was not changed. The expression of pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins but not mRNAs was slightly increased. TCDD-dependent induction of CYP1A1 mRNA and protein was augmented in 50% of clones, but there was no correlation between the CYP1A1 inducibility and expression levels of AhR and HNF4α. Induction of CYP3A4 mRNA by rifampicin was about 1.5-2.5 fold (clones 2, 4, 6, 7) and it was not significantly different from CYP3A4 mRNA induction in parent HepG2. The basal expression of CYP3A4 protein was increased in all clones, but rifampicin-induced expression of CYP3A4 protein was in all clones lower than in parent HepG2. Overall, the stable over-expression of HNF4α in HepG2 cells restores some of the hepatospecific functions, but it has a minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators.

• MeSH
• albuminy metabolismus   MeSH
• alfa-1-antitrypsin metabolismus   MeSH
• antiprotozoální látky farmakologie   MeSH
• cytochrom P-450 CYP1A1 metabolismus   MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• fibrinogen metabolismus   MeSH
• gentamiciny farmakologie   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   MeSH
• hepatocytární jaderný faktor 4 genetika   metabolismus   MeSH
• hepatocyty metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie metabolismus   MeSH
• plazminogen metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• steroidní receptory metabolismus   MeSH
• transfekce MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

TGF-beta is an important mediator of cell growth, differentiation, and proliferation and plays a significant role in both normal and pathological corneal tissue. However, the quantitative relations between TGF-beta1, -beta2 and -beta3 isoforms in human cornea still remain unclear. Therefore, the aim of this study was to determine the gene expression profile of TGF-betas in order to evaluate quantitative relations between the examined transcripts in human corneal epithelium. Transcriptional activity of TGF-beta1, 2, 3, GAPDH and beta-actin genes was estimated on the basis of mRNA copy number per 1 microg of total RNA using the real-time QRT-PCR technique with the SYBR Green I chemistry. Specificity of RT-PCR reaction was confirmed by determination of the characteristic melting temperature for each amplimer. Additionally, the RT-PCR products were separated on 6% polyacrylamide gels and visualized with silver salts. Expression of all TGF-beta genes for the corneal epithelium was determined. Comparable analysis of mRNA copies/1 mug of total RNA for each TGF-beta isoform showed that: TGF-beta1 > TGF-beta2; TGF-beta3 > TGF-beta2; TGF-beta1 = TGF-beta3 (ANOVA test P < 0.0001; post-hoc Tukey's test: TGF-beta1 and TGF-beta2, P = 0.0306; TGF-beta3 and TGF-beta2, P = 0.0045; TGF-beta1 and TGF-beta3 NS). We found different expression of the TGF-beta1, -2 and -3 isoforms in the human corneal epithelium. Such differential expression of TGF-betas suggests that each of them may play a specific role in corneal tissue.

• MeSH
• aktiny genetika   metabolismus   MeSH
• dospělí MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• rohovkový epitel metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transformující růstový faktor beta metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

Neonatal exposure to hyperoxia alters lung development in mice. We tested if retinoic acid (RA) treatment is capable to affect lung development after hyperoxic injury and to maintain structural integrity of lung. The gene of vascular endothelial growth factor A (VEGF-A) is one of the RA-responsive genes. Newborn BALB/c mice were exposed to room air, 40% or 80% hyperoxia for 7 days. One half of animals in each group received 500 mg/kg retinoic acid from day 3 to day 7 of the experiment. At the end of experiment we assessed body weight (BW), lung wet weight (LW), the wet-to-dry lung weight ratio (W/D) and the expression of mRNA for VEGF-A and G3PDH genes. On day 7 the hyperoxia-exposed sham-treated mice (group 80) weighed 20% less than the room air-exposed group, whereas the 80% hyperoxic group treated with RA weighed only 13% less than the normoxic group. W/D values in 80 and 80A groups did not differ, although they both differed from the control group and from 40 groups. There was a significant difference between 40 and 40A groups, but the control group was different from 40 group but not from 40A groups. The 80 and 80A groups had mRNA VEGF-A expression lowered to 64% and 41% of the control group. RA treatment of normoxic and mild hyperoxic groups increased mRNA VEGF-A expression by about 50%. We conclude that the retinoic acid treatment of newborn BALB/c mice exposed for 7 days to 80% hyperoxia reduced the growth retardation in the 80 % hyperoxic group, reduced the W/D ratio in the 40% but not in the 80 % hyperoxic group. Higher VEGF-A mRNA expression in the 80% hyperoxic group treated with RA was not significant compared to the 80% hyperoxic group.

• MeSH
• analýza rozptylu MeSH
• antioxidancia farmakologie   MeSH
• genetická transkripce účinky záření   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   účinky léků   MeSH
• hyperoxie komplikace   metabolismus   patologie   MeSH
• messenger RNA analýza   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• novorozená zvířata MeSH
• plicní alveoly metabolismus   patologie   růst a vývoj   účinky léků   MeSH
• plicní nemoci etiologie   metabolismus   patologie   prevence a kontrola   MeSH
• stupeň závažnosti nemoci MeSH
• tretinoin farmakologie   MeSH
• vaskulární endoteliální růstový faktor A genetika   metabolismus   účinky léků   MeSH
• vývojová regulace genové exprese účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

The flightless bug Pyrrhocoris apterus (L.) is polymorphic for both wing length and flight muscle development. The developed flight muscles of macropterous adults of both sexes first enlarge their volume during the first 5 days after adult emergence, but are then histolyzed in all males and females older than 10 and 14 days, respectively. The flight muscles of brachypterous adult males and females are underdeveloped due to their arrested growth. The total protein content of histolyzed dorsolongitudinal flight muscles from 21-day-old macropterous adults of both sexes is lower than that of developed dorsolongitudinal flight muscles in 5-10-days-old macropterous bugs, but substantially higher than the protein content of underdeveloped dorsolongitudinal flight muscles from adult brachypters. Histolyzed dorsolongitudinal flight muscles differ from the developed ones by decreased quantities of 18 electrophoretically separated proteins. Histolysis of developed dorsolongitudinal flight muscles is accompanied by significant decreases in citrate synthase, glyceraldehyde-3-phosphate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase enzyme activities and an increase in alanine aminotransferase activity, and can be precociously induced by application of a juvenile hormone analogue. This is the first report of flight muscle polymorphism, histolysis of developed flight muscles and its endocrine control in insects displaying non-functional wing polymorphism.

• MeSH
• 3-hydroxyacyl-CoA-dehydrogenasy metabolismus   MeSH
• alanintransaminasa metabolismus   MeSH
• citrátsynthasa metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• financování organizované MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   MeSH
• Heteroptera anatomie a histologie   enzymologie   fyziologie   MeSH
• kosterní svaly anatomie a histologie   enzymologie   fyziologie   MeSH
• křídla zvířecí anatomie a histologie   enzymologie   fyziologie   MeSH
• methopren farmakologie   MeSH
• svalové proteiny fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH

• MeSH
• citrátsynthasa metabolismus   MeSH
• dospělí MeSH
• energetický metabolismus MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hexokinasa metabolismus   MeSH
• hormony štítné žlázy fyziologie   MeSH
• lidé MeSH
• malátdehydrogenasa metabolismus   MeSH
• obezita enzymologie   MeSH
• svaly enzymologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• časové faktory MeSH
• glutamátdehydrogenasa metabolismus   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy metabolismus   MeSH
• hypertrofie enzymologie   etiologie   patofyziologie   MeSH
• kreatinkinasa metabolismus   MeSH
• kur domácí MeSH
• laktátdehydrogenasy metabolismus   MeSH
• lyasy metabolismus   MeSH
• malátdehydrogenasa metabolismus   MeSH
• nemoci svalů etiologie   MeSH
• proteiny metabolismus   MeSH
• svalová kontrakce MeSH
• svaly enzymologie   chirurgie   patofyziologie   patologie   MeSH
• velikost orgánu MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH