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.
- Klíčová slova
- Francisella, glyceraldehyde-3-phosphate dehydrogenase, infection, interacting partners, multitasking, pleiotropy, secretion,
- 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
- Názvy látek
- cytokiny MeSH
- glyceraldehyd-3-fosfátdehydrogenasy MeSH
The Arabidopsis thaliana aldehyde oxidase 3 (AAO3) catalyzes the oxidation of abscisic aldehyde (ABal) to abscisic acid (ABA). Besides ABal, plants generate other aldehydes that can be toxic above a certain threshold. AAO3 knockout mutants (aao3) exhibited earlier senescence but equivalent relative water content compared with wild-type (WT) during normal growth or upon application of UV-C irradiation. Aldehyde profiling in leaves of 24-day-old plants revealed higher accumulation of acrolein, crotonaldehyde, 3Z-hexenal, hexanal and acetaldehyde in aao3 mutants compared with WT leaves. Similarly, higher levels of acrolein, benzaldehyde, crotonaldehyde, propionaldehyde, trans-2-hexenal and acetaldehyde were accumulated in aao3 mutants upon UV-C irradiation. Aldehydes application to plants hastened profuse senescence symptoms and higher accumulation of aldehydes, such as acrolein, benzaldehyde and 4-hydroxy-2-nonenal, in aao3 mutant leaves as compared with WT. The senescence symptoms included greater decrease in chlorophyll content and increase in transcript expression of the early senescence marker genes, Senescence-Related-Gene1, Stay-Green-Protein2 as well as NAC-LIKE, ACTIVATED-BY AP3/P1. Notably, although aao3 had lower ABA content than WT, members of the ABA-responding genes SnRKs were expressed at similar levels in aao3 and WT. Moreover, the other ABA-deficient mutants [aba2 and 9-cis-poxycarotenoid dioxygenase3-2 (nced3-2), that has functional AAO3] exhibited similar aldehydes accumulation and chlorophyll content like WT under normal growth conditions or UV-C irradiation. These results indicate that the absence of AAO3 oxidation activity and not the lower ABA and its associated function is responsible for the earlier senescence symptoms in aao3 mutant.
- Klíčová slova
- Arabidopsis, abscisic acid, aldehyde oxidase, reactive aldehydes, senescence,
- MeSH
- aldehydoxidasa genetika metabolismus MeSH
- aldehydy metabolismus toxicita MeSH
- Arabidopsis genetika fyziologie MeSH
- chlorofyl metabolismus MeSH
- kyselina abscisová metabolismus MeSH
- listy rostlin genetika fyziologie MeSH
- oxidace-redukce MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- senescence rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- AAO3 protein, Arabidopsis MeSH Prohlížeč
- aldehydoxidasa MeSH
- aldehydy MeSH
- chlorofyl MeSH
- kyselina abscisová MeSH
- proteiny huseníčku MeSH
- regulátory růstu rostlin MeSH
Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.
- Klíčová slova
- 1-iminomethyl-2-naphthol, Arabidopsis, Heatin, IAN, NIT1-subfamily, PIF4, aldehyde oxidase, chemical genetics, indole-3-acetonitrile, nitrilases, thermomorphogenesis,
- MeSH
- aldehydoxidasa genetika metabolismus MeSH
- aminohydrolasy genetika metabolismus MeSH
- apomorfin analogy a deriváty farmakologie MeSH
- Arabidopsis účinky léků růst a vývoj MeSH
- herbicidy farmakologie MeSH
- hypokotyl účinky léků růst a vývoj MeSH
- inhibitory enzymů aplikace a dávkování chemie farmakologie MeSH
- kyseliny indoloctové MeSH
- molekulární struktura MeSH
- pikloram farmakologie MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- transkriptom účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 10,11-dihydroxy-N-n-propylnorapomorphine MeSH Prohlížeč
- AAO1 protein, Arabidopsis MeSH Prohlížeč
- aldehydoxidasa MeSH
- aminohydrolasy MeSH
- apomorfin MeSH
- herbicidy MeSH
- inhibitory enzymů MeSH
- kyseliny indoloctové MeSH
- nitrilase MeSH Prohlížeč
- pikloram MeSH
- proteiny huseníčku MeSH
S-nitrosation as a redox-based posttranslational modification of protein cysteine has emerged as an integral part of signaling pathways of nitric oxide across all types of organisms. Protein S-nitrosation status is controlled by two key mechanisms: by direct denitrosation performed by the thioredoxin/thioredoxin reductase system, and in an indirect way mediated by S-nitrosoglutathione reductase (GSNOR). GSNOR, which has been identified as a key component of S-nitrosothiols catabolism, catalyzes an irreversible decomposition of abundant intracellular S-nitrosothiol, S-nitrosoglutathione (GSNO) to oxidized glutathione using reduced NADH cofactor. In plants, GSNOR has been shown to play important roles in plant growth and development and plant responses to abiotic and biotic stress stimuli. In this chapter, optimized protocols of spectrophotometric measurement of GSNOR enzymatic activity and activity staining in native polyacrylamide gels in plant GSNOR are presented.
- Klíčová slova
- Nitric oxide, Plant stress, S-nitrosation, S-nitrosoglutathione reductase, S-nitrosothiols,
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- barvení a značení metody MeSH
- enzymatické testy metody MeSH
- fluorescence MeSH
- NAD chemie MeSH
- nativní elektroforéza na polyakrylamidovém gelu MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- průběh práce MeSH
- rostlinné extrakty izolace a purifikace metabolismus MeSH
- rostliny enzymologie MeSH
- S-nitrosoglutathion chemická syntéza chemie MeSH
- S-nitrosothioly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aldehydoxidoreduktasy MeSH
- formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
- NAD MeSH
- oxid dusnatý MeSH
- rostlinné extrakty MeSH
- S-nitrosoglutathion MeSH
- S-nitrosothioly MeSH
BACKGROUND: ALDH-2 has been considered an important molecular target for the treatment of drug addiction due to its involvement in the metabolism of the neurotransmitter dopamine: however, the molecular basis for the selective inhibition of ALDH-2 versus ALDH-1 should be better investigated to enable a more pragmatic approach to the design of novel ALDH-2 selective inhibitors. OBJECTIVE: In the present study, we investigated the molecular basis for the selective inhibition of ALDH-2 by the antioxidant isoflavonoid daidzin (IC50 = 0.15 μM) compared to isoform 1 of ALDH through molecular dynamics studies and semiempirical calculations of the enthalpy of interaction. METHODS: The applied methodology consisted of performing the molecular docking of daidzin in the structures of ALDH-1 and ALDH-2 and submitting the lower energy complexes obtained to semiempirical calculations and dynamic molecular simulations. RESULTS: Daidzin in complex with ALDH-2 presented directed and more specific interactions, resulting in stronger bonds in energetic terms and, therefore, in enthalpic gain. Moreover, the hydrophobic subunits of daidzin, in a conformationally more restricted environment (such as the catalytic site of ALDH-2), promote the better organization of the water molecules when immersed in the solvent, also resulting in an entropic gain. CONCLUSION: The molecular basis of selective inhibition of ALDH-2 by isoflavonoids and related compounds could be related to a more favorable equilibrium relationship between enthalpic and entropic features. The results described herein expand the available knowledge regarding the physiopathological and therapeutic mechanisms associated with drug addiction.
- Klíčová slova
- ALDH-2, daidzin, isoflavonoids, molecular dynamics, nucleus accumbens, selective inhibition,
- MeSH
- aldehyddehydrogenasa metabolismus MeSH
- dopamin metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- isoflavony farmakologie MeSH
- poruchy spojené s užíváním psychoaktivních látek farmakoterapie MeSH
- simulace molekulového dockingu MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aldehyddehydrogenasa MeSH
- daidzin MeSH Prohlížeč
- dopamin MeSH
- inhibitory enzymů MeSH
- isoflavony MeSH
Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we designed two sets of primers for the detection and quantification of the multicopper oxidase gene (MCO), which encodes an enzyme involved in BAs degradation, and endogenous (glyceraldehyde-3-phosphate dehydrogenase) gene (GAPDH) in Lactobacillus casei group by real-time PCR (qPCR). We tested 15 Lactobacillus strains in the screening assays (thus, MCO gene possessing assay (PCR) and monitoring of BAs degradation by HPLC-UV), in which Lactobacillus casei CCDM 198 exhibited the best degradation abilities. For this strain, we monitored the expression of the target gene (MCO) in time (qPCR), the effect of redox treatments (cysteine, ascorbic acid) on the expression of the gene, and the ability to degrade BAs not only in a modified MRS medium (MRS/2) but also in a real food sample (milk). Moreover, decarboxylase activity (ability to form BAs) of this strain was excluded. According to the results, CCDM 198 significantly (P < 0.05) reduced BAs (putrescine, histamine, tyramine, cadaverine), up to 25% decline in 48 h. The highest level of relative expression of MCO (5.21 ± 0.14) was achieved in MRS/2 media with cysteine.
- Klíčová slova
- Biogenic amines degradation, Histamine, Lactobacillus casei, Primers, qPCR,
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biogenní aminy analýza metabolismus MeSH
- cystein analýza metabolismus MeSH
- glyceraldehyd-3-fosfátdehydrogenasy genetika MeSH
- kultivační média chemie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- kyselina askorbová analýza metabolismus MeSH
- Lactobacillus casei enzymologie genetika růst a vývoj metabolismus MeSH
- Lactobacillus enzymologie genetika růst a vývoj metabolismus MeSH
- mléko chemie MeSH
- oxidoreduktasy genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- bakteriální proteiny MeSH
- biogenní aminy MeSH
- cystein MeSH
- glyceraldehyd-3-fosfátdehydrogenasy MeSH
- kultivační média MeSH
- kyselina askorbová MeSH
- oxidoreduktasy MeSH
Nitric oxide plays an important role in the pathogenesis of Pseudoidium neolycopersici, the causative agent of tomato powdery mildew. S-nitrosoglutathione reductase, the key enzyme of S-nitrosothiol homeostasis, was investigated during plant development and following infection in three genotypes of Solanum spp. differing in their resistance to P. neolycopersici. Levels and localization of reactive nitrogen species (RNS) including NO, S-nitrosoglutathione (GSNO) and peroxynitrite were studied together with protein nitration and the activity of nitrate reductase (NR). GSNOR expression profiles and enzyme activities were modulated during plant development and important differences among Solanum spp. genotypes were observed, accompanied by modulation of NO, GSNO, peroxynitrite and nitrated proteins levels. GSNOR was down-regulated in infected plants, with exception of resistant S. habrochaites early after inoculation. Modulations of GSNOR activities in response to pathogen infection were found also on the systemic level in leaves above and below the inoculation site. Infection strongly increased NR activity and gene expression in resistant S. habrochaites in contrast to susceptible S. lycopersicum. Obtained data confirm the key role of GSNOR and modulations of RNS during plant development under normal conditions and point to their involvement in molecular mechanisms of tomato responses to biotrophic pathogens on local and systemic levels.
- Klíčová slova
- Nitric oxide, Powdery mildew, Pseudoidium neolycopersici, Reactive nitrogen species, S-nitrosoglutathione reductase, Solanum spp., Tomato,
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- Ascomycota patogenita MeSH
- genotyp MeSH
- nemoci rostlin * mikrobiologie MeSH
- reaktivní formy dusíku metabolismus MeSH
- Solanum lycopersicum enzymologie mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aldehydoxidoreduktasy MeSH
- formaldehyde dehydrogenase, glutathione-independent MeSH Prohlížeč
- reaktivní formy dusíku MeSH
Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.
- Klíčová slova
- aldehyde oxidase, capillary electrophoresis, enzyme immobilization, magnetic nanoparticles, micellar electrokinetic chromatography,
- MeSH
- aldehydoxidasa analýza metabolismus MeSH
- elektroforéza kapilární MeSH
- enzymatické testy * MeSH
- enzymy imobilizované analýza metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aldehydoxidasa MeSH
- enzymy imobilizované MeSH
Conditional gene targeting in mice by means of Cre-loxP strategy represents a powerful approach to study mammalian gene function. This approach is however dependent on the availability of suitable strains of mice with a tissue or time restricted activity of the Cre recombinase. Here we describe Aldh3-Cre transgenic mice as a useful tool to conditionally delete genes in cornea, a specialized transparent tissue found on the anterior-most part of the eye, which acts as a protective barrier and contributes to the refractive power. Using a set of floxed alleles we demonstrate high Aldh3-Cre activity in corneal epithelial cells, corneal stroma and conjunctival epithelial cells at postnatal stages. Aldh3-Cre will thus be particularly beneficial for functional analysis of genes which are vital for postnatal development of cornea and conjunctiva.
- MeSH
- aldehyddehydrogenasa genetika MeSH
- alely MeSH
- delece genu MeSH
- epitelové buňky fyziologie MeSH
- genový targeting metody MeSH
- integrasy genetika MeSH
- konjunktiva fyziologie MeSH
- myši transgenní genetika fyziologie MeSH
- myši MeSH
- rohovka fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aldehyddehydrogenasa MeSH
- Aldh3a1protein, mouse MeSH Prohlížeč
- Cre recombinase MeSH Prohlížeč
- integrasy MeSH
OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.
- Klíčová slova
- Japanese, genome-wide association study (GWAS), gout/hyperuricaemia, selection pressure analysis, subtype specific locus,
- MeSH
- ABC transportér z rodiny G, člen 2 genetika MeSH
- celogenomová asociační studie * MeSH
- dna (nemoc) epidemiologie genetika MeSH
- fenotyp MeSH
- genetická predispozice k nemoci etnologie MeSH
- genetické lokusy MeSH
- genotyp MeSH
- hodnocení rizik MeSH
- incidence MeSH
- lidé MeSH
- mitochondriální aldehyddehydrogenasa genetika MeSH
- nádorové proteiny genetika MeSH
- prognóza MeSH
- referenční hodnoty MeSH
- studie případů a kontrol MeSH
- stupeň závažnosti nemoci MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- metaanalýza MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Japonsko MeSH
- Názvy látek
- ABC transportér z rodiny G, člen 2 MeSH
- ABCG2 protein, human MeSH Prohlížeč
- ALDH2 protein, human MeSH Prohlížeč
- mitochondriální aldehyddehydrogenasa MeSH
- nádorové proteiny MeSH
