Jacek Skała

Monitoring changes in anthocyanin and steroid alkaloid glycoside content in lines of transgenic potato plants using liquid chromatography/mass spectrometry

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection.

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.

Biological activity and structure dependent properties of cuprous iodide complexes with phenanthrolines and water soluble tris (aminomethyl) phosphanes.

This paper presents the biological activity of copper(I) iodide complexes with 1,10-phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (dmp) and three tris (aminomethyl) phosphanes: P(CH2N(CH2CH2)2NCH3)3 (1), P(CH2N(CH2CH2)2O)3 (2) and P (CH2N(CH3)CH2CH2OH)3 (3). Crystallographic and DFT data indicate a significantly stronger binding ability of 3 in the complexes [CuI (phen) P (CH2N (CH3)CH2CH2OH)3] (3P) and [CuI(dmp)P(CH2N(CH3)CH2CH2OH)3] (3N) in comparison to the 1 or 2 ligands. Most probably, this is caused by the relatively small steric requirements of 3. The complexes with dmp exhibit a very high in vitro activity against the Staphylococcus aureus strain (MIC - minimal inhibitory concentration: 2.5-5 μg/mL) and Candida albicans diploid fungus (MIC: 1.25-2.5 μg/mL). All the tested complexes also show a strong in vitro antitumor activity against human ovarian carcinoma cell lines: MDAH 2774 (IC50: 7-2 μM) and cisplatin-resistant SCOV3 (IC50: 3-2 μM). Interestingly, the complexes with dmp of higher biological activity more weakly interact with bovine serum albumin (BSA) and less efficiently cleave the pBluescriptSK+ plasmid.

Genes of phenylpropanoid pathway are activated in early response to Fusarium attack in flax plants.

Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant death. To protect itself, the plant activates a number of genes and metabolic pathways, both to counteract the effects of the pathogen, and to eliminate the threat. The identification of the plant genes which respond to infection is the approach, that has been used in this study. Forty-seven flax genes have been identified by means of cDNA subtraction method as those, which respond to pathogen infection. Subtracted genes were classified into several classes and the prevalence of the genes involved in the broad spectrum of antioxidants biosynthesis has been noticed. By means of semi-quantitative RT-PCR and metabolite profiling, the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively verified. We identified the key genes of the synthesis of these compounds. At the same time we determined the level of the metabolites produced in the phenylpropanoid pathway (flavonoids, phenolic acids) in early response to Fusarium attack by means of GC-MS technique. To the best of our knowledge this is the first report to describe genes and metabolites of early flax response to pathogens studied in a comprehensive way.

Interaction of aminoglycosides and their copper(II) complexes with nucleic acids: implication to the toxicity of these drugs

Cupric complexes of eight aminoglycosidic antibiotics were screened for their specific behavior towards tRNAPhe, both in oxidative and neutral surrounding. Without H2O2, the cleavage efficiency was dependent on the resultant charge of the molecule. A comparative assay using tRNAPhe devoid of the natural hypermodification in the anticodon loop proved that hypermodification is indispensable for site recognition and subsequent cleavage. The intensity of single and double strand scissions in plasmid DNA also proceeded in a charge-dependent manner. Unlike free antibiotics, their cupric complexes in the presence of H2O2, facilitated plasmid linearisation and degradation. The participation of ROS in those processes was confirmed using NDMA as a reporter molecule, whose consumption was influenced by the protonation state of the complex.

Repression of six 14-3-3 protein isoforms resulting in the activation of nitrate and carbon fixation key enzymes from transgenic potato plants

Six 14-3-3 full-size cDNAs from a potato plant were cloned and sequenced, and their high-sequence homology was established. The expression profile revealed developmental regulation and lack of organ specificity of 14-3-3 isoform synthesis in potato leaves. In order to analyse their function, transgenic plants with repression of all found isoforms were created. Since, 14-3-3 regulates in vitro assay sucrose phosphate synthase (SPS) and nitrate reductase (NR) activities, both enzyme activities and products of the interacting enzyme were analysed for transgenic plants. It was found that all transgenic lines showed significant increase in SPS and NR activities. The increase in enzyme activities was complemented by the addition of recombinant 14-3-3 protein either from potato or C. pepo. Thus, it is suggested that 14-3-3 is the main regulator of NR and SPS activities in in vivo trials and the regulatory function is isoform-independent. Sucrose and glutamate contents corresponded with the enzyme activity increase especially in case of two transgenic lines, G3.40 and G3.49, respectively. Four out of five analysed transgenic lines showed significant increase in tuber starch accumulation.

Bactericidal, opsonophagocytic and anti-adhesive effectiveness of cross-reactive antibodies against Moraxella catarrhalis

Moraxella catarrhalis is a human-restricted significant respiratory tract pathogen. The bacteria accounts for 15-20% of cases of otitis media in children and is an important causative agent of infectious exacerbations of chronic obstructive pulmonary disease in adults. The acquisition of new M. catarrhalis strains plays a central role in the pathogenesis of both mentioned disorders. The antibody-dependent immune response to this pathogen is critical for its effective elimination. Thus, the knowledge about the protective threshold of cross-reactive antibodies with defined functionality seems to be important. The complex analysis of broad-spectrum effectiveness of cross-reactive antibodies against M. catarrhalis has never been performed. The goal of the present study was to demonstrate and compare the bactericidal, opsonophagocytic and blocking function of cross-reacting antibodies produced in response to this bacterium or purified outer membrane proteins incorporated in Zwittergent-based micelles. The multivalent immunogens were used in order to better mimic the natural response of the host. The demonstrated broad-spectrum effectiveness of cross-reactive antibodies in pathogen eradication or inhibition strongly indicates that this pool of antibodies by recognition of pivotal shared M. catarrhalis surface epitopes seems to be an essential additional source to control host-microbe interaction.

DNA Oxidative Cleavage Induced by the Novel Peptide Derivatives of 3-(quinoxalin-6-yl)alanine in Combination with Cu(II) or Fe(II) Ions

Three model dipeptides containing 3-(2,3-di(pyridin-2-yl)quinoxalin-6-yl)alanine, 3-(dipyrido[3,2-a:2,3-c]phenazin-11-yl)alanine, and 3-(2,3-diphenylquinoxalin-6-yl)alanine were studied with respect to their ability to bind selected transition metal ions, such as Cu(II), Fe(II), Ni(II), Co(II), Mn(II), and Cr(III). It was found that only Cu(II) and Fe(II) ions could form stable complex species with the studied compounds. The ability to form the complexes correlated well with DNA damage experiments. Only the ferrous and cupric complexes are capable of generating both single- and double-strand scissions. However, double-strand breakages appear to be dominating lesions in the presence of hydrogen peroxide, especially for copper(II) containing systems. The quantity of breakage products in the presence of N-(3-(dipyrido[3,2-a:2,3-c]phenazine-11-yl)alanyl)glycine complexes was the highest as compared to the complexes of the remaining compounds. Moreover, this ligand was the only one that cleaved DNA in the absence of either Cu(II) or Fe(II) ions.

Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils

Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) play both pro- and anti-inflammatory activities and are produced during infection and inflammation. Moraxella catarrhalis is one of the leading infectious agents responsible for inflammatory exacerbation in chronic obstructive pulmonary disease (COPD). Since the airway inflammation in COPD is connected with activation of both epithelial cells and accumulated neutrophils, in this study we determined the in vitro effects of neuropeptides on the inflammatory potential of these cells in response to M. catarrhalis outer membrane vesicle (OMV) stimulant. The various OMV-mediated proinflammatory effects were demonstrated. Next, using hBD-2-pGL4[luc2] plasmid with luciferase reporter gene, SP and CGRP were shown to inhibit the IL-1β-dependent expression of potent neutrophil chemoattractant, hBD-2 defensin, in transfected A549 epithelial cells (type II alveolar cells) upon OMV stimulation. Both neuropeptides exerted antiapoptotic activity through rescuing a significant fraction of A549 cells from OMV-induced cell death and apoptosis. Finally, CGRP caused an impairment of specific but not azurophilic granule exocytosis from neutrophils as shown by evaluation of gelatinase-associated lipocalin (NGAL) or CD66b expression and elastase release, respectively. Concluding, these findings suggest that SP and CGRP mediate the dampening of proinflammatory action triggered by M. catarrhalis OMVs towards cells engaged in lung inflammation in vitro.