The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii.

MAIN CONCLUSION: Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

Mutations in Selected ABA-Related Genes Reduce Level of Arabidopsis thaliana Susceptibility to the Beet Cyst Nematode Heterodera schachtii.

Heterodera schachtii is a common parasite of many important crops such as beets and Brassicaceae (oilseed rape, cabbage or mustard). Arabidopsis thaliana is a model plant also used for studying defence responses to pathogens or pest infections. Defence responses of plants are often regulated and fine-tuned by stress phytohormones: salicylic acid (SA), jasmonic acid (JA), ethylene (Et) and abscisic acid (ABA), of which the role of ABA in these responses is the least examined. The aim of this study was to show, if and which genes related to ABA turnover can be modulated during the development of nematode-induced feeding sites in A. thaliana roots. To answer the question, we performed infection tests on wild type and ABA mutant roots and analysed the expression levels of selected ABA-related genes (ABI1, ABI2, ABI5, PYL5, PYL6, CYP707A1 and CYP707A4) at the early stage of root infection. Our results show that the expression of ABI2, ABI5 (ABA signalling) and CYP707A4 (ABA metabolism) genes was upregulated in feeding sites at 4 dpi, whereas the level of expression of PYL5 and PYL6 (ABA receptors) genes was decreased. Mutations in ABI1, ABI2, ABI5, CYP707A1 or CYP707A4 genes led to a decrease of A. thaliana susceptibility verbalised as the number of fully developed females, whereas mutations in PYL5 or PYL6 genes did not influence the number of females of the nematode. Based on the results, it can be concluded that the modifications of analysed ABA-related gene expression are required for the proper development of nematodes; however, further in-depth analyses are required.

Arabidopsis thaliana AtHRS1 gene is involved in the response to Heterodera schachtii infection and its overexpression hampers development of syncytia and involves a jasmonic acid-dependent mechanism.

Sedentary plant parasitic nematodes have developed competences to reprogram host plant cell metabolism via sophisticated manipulation of gene expression, leading to the formation of permanent feeding sites for an unlimited source of food. Arabidopsis thaliana and the beet cyst nematode Heterodera schachtii is a good model for studying the mechanisms of compatible plant-nematode interactions and basic plant responses to nematode infection. Transcription factors are proteins that modulate plant reactions during regular development and under different biotic and abiotic stresses via direct binding to promoter regions of genes. Here, we report on the AtHRS1 gene encoding a MYB-related transcription factor belonging to the GARP family, whose expression is downregulated in syncytia, as confirmed by gene expression analysis. Constitutive overexpression of AtHRS1 disturbed the development of nematode-induced syncytia and led to a reduction in the number of developed females in transgenic A. thaliana roots. In contrast, the hrs1 mutant with decreased expression of AtHRS1 was more susceptible to cyst nematode infection. The influence of AtHRS1 on selected elements of the JA-dependent defence pathway suggests its mode of action in plant response to nematode attack. Based on these results, we suggest that the downregulation of AtHRS1 expression by nematode is important for its successful development.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia.

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants' responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

The Interactions of Nintedanib and Oral Anticoagulants-Molecular Mechanisms and Clinical Implications.

Nintedanib is a synthetic orally active tyrosine kinase inhibitor, whose main action is to inhibit the receptors of the platelet-derived growth factor, fibroblast growth factor and vascular endothelial growth factor families. The drug also affects other kinases, including Src, Flt-3, LCK, LYN. Nintedanib is used in the treatment of idiopathic pulmonary fibrosis, chronic fibrosing interstitial lung diseases and lung cancer. The mechanism of action suggests that nintedanib should be considered one of the potential agents for inhibiting and revising the fibrosis process related to COVID-19 infections. Due to the known induction of coagulation pathways during COVID-19 infections, possible interaction between nintedanib and anticoagulant seems to be an extremely important issue. In theory, nintedanib could increase the bleeding risk, thrombosis and lead to thrombocytopenia. The data from clinical trials on the concomitant use of nintedanib and antithrombotic agents is very limited as this patient group was within the standard exclusion criteria. Nintedanib is an important therapeutic option, despite its interaction with anticoagulants. If anticoagulant therapy is necessary, the more effective and safer option is the concomitant administration of DOACs and nintedanib, especially when drug-monitored therapy will be used in patients at high risk of bleeding complications.

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

Diminished toxicity of C-1748, 4-methyl-9-hydroxyethylamino-1-nitroacridine, compared with its demethyl analog, C-857, corresponds to its resistance to metabolism in HepG2 cells.

The narrow "therapeutic window" of anti-tumour therapy may be the result of drug metabolism leading to the activation or detoxification of antitumour agents. The aim of this work is to examine (i) whether the diminished toxicity of a potent antitumour drug, C-1748, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine, compared with its 4-demethyl analogue, C-857, results from the differences between the metabolic pathways for the two compounds and (ii) the impact of reducing and/or hypoxic conditions on studied metabolism. We investigated the metabolites of C-1748 and C-857 formed in rat and human liver microsomes, with human P450 reductase (POR) and in HepG2 cells under normoxia and hypoxia. The elimination rate of C-1748 from POR knockout mice (HRN) was also evaluated. Three products, 1-amino-9-hydroxyethylaminoacridine, 1-aminoacridinone and a compound with an additional 6-membered ring, were identified for C-1748 and C-857 in all studied metabolic systems. The new metabolite was found in HepG2 cells. We showed that metabolic rate and the reactivity of metabolites of C-1748 were considerably lower than those of C-857, in all investigated metabolic models. Compared with metabolism under normoxia, cellular metabolism under hypoxia led to higher levels of 1-aminoacridine and aza-acridine derivatives of both compounds and of the 6-membered ring metabolite of C-1748. In conclusion, the crucial role of hypoxic conditions and the direct involvement of POR in the metabolism of both compounds were demonstrated. Compared with C-857, the low reactivity of C-1748 and the stability of its metabolites are postulated to contribute significantly to the diminished toxicity of this compound observed in animals.

Oral versus intravenous hydration and renal function in diabetic patients undergoing percutaneous coronary interventions.

BACKGROUND: Contrast-induced nephropathy (CIN) is a serious complication of percutenous coronary interventions (PCI). Proper hydration reduces the risk of PCI. Wheter oral hydration is as effective as intravenous one has not been well established. AIM: To determine the effects of oral hydration with mineral water versus intravenous hydration with isotonic solution (0.9% NaCl) on renal function in diabetic patients undergoing coronary angiography and angioplasty. METHODS: The study included 102 patients (age 67 ± 7.8 years, 44 female/58 male). Eligible patients (group 1 - 52 pts) were hydrated intravenously (1 mL/kg/h) 6 hours before and during 12 hours following PCI with isotonic solution (0.9% NaCl). Fifty patients (group 2) were randomised to receive oral mineral water (1 mL/kg/h) 6-12 hours before and during 12 hours following angiography or angioplasty. All patients during the procedure received contrast medium ioversol. Primary endpoint of the study was the evaluation of renal function before and 72 hours after contrast medium administration. RESULTS: Baseline creatinine clearance was 70.3 ± 21.22 mL/min in group 1 and 78.69 ± 19.92 mL/min in group 2 (NS). The mean volume of contrast medium was 101.1 ± 36.7 mL in group 1 and 110.4 ± 45.3 mL in group 2 (NS). At 72 hours after the procedure, creatinine clearance was 65.3 ± 23.39 mL/min in group 1 and 73.5 ± 21.94 mL/min in group 2 (NS). CONCLUSIONS: Our study demonstrates that the oral hydration with mineral water and intravenous hydration with 0.9% NaCl have similar effects on renal function in diabetic patients undergoing coronary angiography and angioplasty.

[Cytochrome P450 isoenzymes in metabolism of endo- and exogenic compounds]. / Izoenzymy cytochromu P450 w metabolizmie zwiazków endo- i egzogennych.

Cytochrome P450 enzymes (CYPs) belong to hemeproteins found in all living organisms. In eucaryotic cells they are responsible for biosynthesis and transformations of endogenic lipids as well as for the metabolism of xenobiotics, including therapeutic agents. C-Oxidation (hydroxylation, epoxydation, peroxydation), N-oxidation and S-oxidation as well as oxidative O-, S-, and N-dealkylation of substrates are catalysed by CYPs. These monooxygenation reactions sometimes result in dimerisation, isomerisation or cyclisation of the substrate. Human cytochrome P450 isoenzymes are described by 57 genes and products of their expression are different in specificity. For instance CYP51A1 is crucial for sterol biosynthesis, whereas CYP7A1, 7B1 and 39A1 take part in synthesis of bile acids and CYP46A1 in metabolism of cholesterol. Therapeutic agents are metabolised mainly by CYP3A4, 2D6, 2C9 and 2C19. In addition, CYP2E1 takes part in metabolism of ethyl alcohol and CYP1A1/2 in activation of carcinogens. Metabolism of xenobiotics seems to be the defence mechanism against toxic effects of strange chemicals, whereas, it is also the way of drug activation and detoxication.

[NADPH-cytochrome P450 reductase, not only the partner of cytochrome P450]. / Reduktaza NADPH: cytochrom P450--nie tylko partner cytochromu P450.

NADPH-cytochrome P450 reductase, CPR, the enzyme of the majority of eucaryotic cells belongs to the family of diflavin reductases and is usually located in endoplasmic reticulum. This protein is build of three domains. The first one, C-terminal, binds FAD and NADPH, the second one, N-terminal, binds FMM, whereas the third one is the regulatory domain. Catalytic cycle of the enzyme runs by intermediate FMNH-FADH with the participation of conformational changes induced by NADPH binding to the active centre of the enzyme. It has been shown in mice that CPR was necessary for the action of cytochrome P450 monooxygenase system, but this system is not crucial for animal surviving. CPR participates also in electron transport to cytochrome b5, heme oxidase, squalen monooxygenase and 7-dehydrocholesterole reductase. Furthermore, its own crucial task is the catalysis of reductive metabolism of prodrugs, particularly antitumor agents.

[Polymorphism and the level of P450 gene expression in xenobiotic metabolism]. / Rola polimorfizmu i zróznicowanej ekspresji genów cytochromów P450 w metabolizmie ksenobiotyków.

Polymorphism and the level of P450 gene expression influence metabolic pathway of xenobiotics. The highest number, over 40 polymorphic forms were determined for CYP2D6 enzyme and they expressed various activity. Whereas, only two forms of lower activity were found in the case of CYP2C9. However, CYP3A4 mutations have not influenced metabolic activity of the enzyme. Three transcription factors PXR, CAR and AhR are crucial for gene expression regulation of cytochrome P450. They regulate the induction of CYP3A4, CYP2B6 and CYP1A2 genes, respectively. PXR is activated by binding specific ligand, whereas the activator of constitutive CAR receptor seems not to be the ligand and it might induce dephosphorylation. After being transported to the nucleus, activated receptors PXR, CAR or AhR formed heterodimers and then, with participation of the following coactivators bind to PBREM, XREM or DRE regulatory motifs of DNA, respectively. The activation of nuclear receptors strongly influences the drug-drug interactions that are essential for the efficiency of drug, particularly in the field of anticancer therapy.

Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver.

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.

Tissue-culture-responsive and autotetraploidy-responsive changes in metabolic profiles of cucumber (Cucumis sativus L.).

Somaclonal variation commonly occurs during in vitro plant regeneration and may introduce unintended changes in numerous plant characters. In order to assess the range of tissue-culture-responsive changes on the biochemical level, the metabolic profiles of diploid and tetraploid cucumber R1 plants regenerated from leaf-derived callus were determined. Gas chromatography and mass spectrometry were used for monitoring of 48 metabolites and many significant changes were found in metabolic profiles of these plants as compared to a seed-derived control. Most of the changes were common to diploids and tetraploids and were effects of tissue culture. However, tetraploids showed quantitative changes in 14 metabolites, as compared to regenerated diploids. These changes include increases in serine, glucose-6P, fructose-6P, oleic acid and shikimic acid levels. Basing on this study we conclude that the variation in metabolic profiles does not correlate directly with the range of genome changes in tetraploids.