N9-Substituted N⁶-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N(6)-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2-C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N(6)-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N(6)-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by (1)H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ≥ 50 μM.     

Effects of cyclosporine A and its immunosuppressive or non-immunosuppressive derivatives [D-Ser]8-CsA and Cs9 on mitochondria from different brain regions

We studied the functional properties of isolated brain mitochondria (BM) prepared from total rat brain (BM(total)) or from cerebral subregions under basal and Ca(2+) overload conditions in order to evaluate the effects of cyclosporine A (CsA) in a regiospecific manner. CsA-induced effects were compared with those of two derivatives-the none-immunosuppressive [O-(NH(2)(CH2)(5)NHC(O)CH(2))-D-Ser](8)-CsA (Cs9) and its congener, the immunosuppressive [D-Ser](8)-CsA. The glutamate/malate-dependent state 3 respiration of mitochondria (state 3(glu/mal)) differed in region-specific manner (cortex > striatum = cerebellum > substantia nigra > hippocampus), but was significantly increased by 1μM CsA (+21±5%) in all regions. Ca(2+) overload induced by addition of 20μM Ca(2+) caused a significant decrease of state 3(glu/mal) (-45 to -55%) which was almost completely prevented in the presence of 1μM CsA, 1μM Cs9 or 1μM [D-Ser](8)-CsA. Mitochondrial Ca(2+) accumulation thresholds linked to permeability transition (PT) as well as the rate and completeness of mitochondrial Ca(2+) accumulation differed between different brain regions. For the first time, we provide a detailed, regiospecific analysis of Ca(2+)-dependent properties of brain mitochondria. Regardless of their immunosuppressive impact, CsA and its analogues improved mitochondrial functional properties under control conditions. They also preserved brain mitochondria against Ca(2+) overload-mediated PT and functional impairments. Since Cs9 does not mediate immunosuppression, it might be used as a more specific PT inhibitor than CsA.     

Tools and methodologies to support more sustainable biofuel feedstock production

Increasingly, government regulations, voluntary standards, and company guidelines require that biofuel production complies with sustainability criteria. For some stakeholders, however, compliance with these criteria may seem complex, costly, or unfeasible. What exiting tools, then, might facilitate compliance with a variety of biofuel-related sustainability criteria? This paper presents four existing tools and methodologies that can help stakeholders assess (and mitigate) potential risks associated with feedstock production, and can thus facilitate compliance with requirements under different requirement systems. These include the Integrated Biodiversity Assessment Tool (IBAT), the ARtificial Intelligence for Ecosystem Services (ARIES) tool, the Responsible Cultivation Areas (RCA) methodology, and the related Biofuels + Forest Carbon (Biofuel + FC) methodology.     

Biochemical responses and oxidative stress in <Emphasis Type="Italic">Francisella tularensis</Emphasis> infection: a European brown hare model

Background  

The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections.     

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (T_m) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.     

Elite young soccer players have smaller inter-limb asymmetry and better body composition than non-elite players

Body composition (BC) and inter-limb anthropometric asymmetries (LA) may influence the physical performance of soccer players. This study aimed to determine differences in BC and LA among soccer across four performance levels. The study involved 110 male soccer players participating in Czech senior teams who were grouped into four different performance levels (i.e. G1: national team, G2: 1^st division, G3: 2^nd division, G4: 3^rd division). The following BC and LA parameters were compared among groups: body height, body mass, absolute fat-free mass, relative fat-free mass (FFMrel), percentage of fat mass (FM), total body water (TBW), intracellular water (ICW), extracellular water (ECW), phase angle, and bilateral muscle mass differences in the upper and lower extremities. Significant differences were observed in BC parameters among all groups (λ = 0.06, F75,246 = 5.38, p = 0.01, η_p² = 0.62). High-performance players (i.e. G1, G2) had significantly (p < 0.01) lower FM than lower performance players (i.e. G3, G4). The lowest values of FFMrel, relative TBW, relative ICW and ECW were detected in the lowest-performance players (i.e. G4). Significantly lower bilateral muscle mass differences were detected in G1 players (2.71 ± 1.26%; p < 0.01) compared with G4 players (3.95 ± 1.17%). G1 and G2 players had a higher proportion of muscle mass in the torso (p < 0.01) and upper limbs than G3 and G4 (p < 0.01). Elite and high-performance players have better BC and lower inter-limb anthropometric asymmetries compared with low-performance level players.