Spatial arrangement and genetic structure in Gentianella aspera in a regional, local, and temporal context

Gentianella aspera is a biennial plant of various nutrient-poor grasslands that has become rare in the landscapes outside the Alps of eastern Austria. Using AFLP fingerprinting we investigated: (1) effects of spatial structure on genetic structure in a large vineyard population that is confined to the embankments separating the grapevines; (2) temporal variation in genetic diversity and structure in this population; (3) relationships with other populations in a regional context. On the regional scale, moderate isolation by distance among populations was revealed by a Mantel test. Bayesian analysis of population structure indicated three spatially distinct gene pools and an additional one within the vineyard population. Within this population, spatial autocorrelation analysis revealed a positive correlation between genetic and spatial distance up to 50 m. Patterns found by PCoA were not in line with a priori defined subpopulations and indicated substantial gene flow across embankments. AMOVA revealed low differentiation among both the subpopulations that were found on the linear embankments and among two local groups of these subpopulations. We found, however, striking differences in the among-group variation between the 2 years, i.e., between two local groups within the generations and between those groups among generations. This was due to the highly variable larger group of the younger generation, in which an additional gene pool was identified by Bayesian analysis of population structure. Based on these results we discuss scenarios of local and regional dynamics within and among G. aspera populations.     

Synthesis and antiprotozoal activity of 2,5-bis[amidinoaryl]thiazoles

Seven novel diamidino 2,5-bis(aryl)thiazoles (5a–g) were synthesized and evaluated against Trypanosoma brucei rhodensiense (T. b. r.) and Plasmodium falciparum (P. f.). The diamidines were obtained directly from the corresponding bis-nitriles (4a–g) by the action of lithium bis(trimethylsilyl)amide. The bis-nitriles 4a–f were synthesized in four steps starting with the Stille coupling of 2-tributyltinthiazole with the appropriate cyanoaryl halide. The bis-nitrile 5g was obtained by the palladium facilitated coupling of the mixed tin-silyl reagent 2-trimethylsilyl-5-trimethyltinthiazole with 2-bromo-5-cyanopyridine. The amidoxime potential prodrugs 6a–e, 6g were obtained by the reaction of hydroxylamine with the bis-nitriles. O-Methylation of the amidoximes gave the corresponding N-methoxyamidines 7a–c, 7e, 7g. The diamidines showed strong DNA binding affinity as reflected by ΔT_m measurements. Four of the diamidines 5a, 5b, 5d and 5e were highly active in vitro against P. f. giving IC₅₀ values between 1.1 and 2.5 nM. The same four diamidines showed IC₅₀ values between 4 and 6 nM against T. b. r. The selectivity indices ranged from 233 to 9175. One diamidine 5a produced one of four cures at an ip dose of 4 × 5 mg/kg in the STIB900 mouse model for acute African trypanosomiasis. The amidoxime and N-methoxyamidine of 5a were the only produgs to provide cures (1/4 cures) in the same mouse model on oral dosage at 4 × 25 mg/kg.     

Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Sirtuins catalyze the NAD⁺ dependent deacetylation of N^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP-ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε-alkanoyl, N^ε-α,β-unsaturated carbonyl and N^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.     

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions

Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 μg mL⁻¹ arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 μg mL⁻¹ As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 μg mL⁻¹.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.     

Adiponectin and negative mood in healthy premenopausal and postmenopausal women

Negative mood and stress are associated with cardiovascular and metabolic disease. There are likely many physiological mechanisms underlying the poor health outcomes. The relationship of psychological states (negative mood, life stress, and stress-responsive hormones) and adiponectin, an adipokine that promotes insulin sensitivity, was investigated in two separate studies. The two groups of participants included 52 healthy, premenopausal women, and 63 postmenopausal women with a range of stress levels. The relationship between adiponectin and psychological state (perceived stress and negative mood) was examined cross-sectionally in both groups of participants, but also prospectively (1 year later) in the group of postmenopausal women.In premenopausal women, negative mood and nocturnal urinary epinephrine were significantly related to adiponectin, independent of BMI. In postmenopausal women, negative mood was not associated with adiponectin cross-sectionally, but negative mood was a significant predictor for lower levels of adiponectin 1 year later, independent of initial adiponectin concentrations and changes in body mass index. Lastly, having a depressive disorder was related to lower adiponectin. As adiponectin levels are associated with insulin resistance, obesity, and diabetes mellitus, these findings suggest there may be an adiponectin-mediated pathway explaining in part how negative mood affects metabolic health. Mechanistic studies are needed to explore this potential relationship further.     

FGFR4 GLY388 isotype suppresses motility of MDA-MB-231 breast cancer cells by EDG-2 gene repression

Clinical investigations of an FGFR4 germline polymorphism, resulting in substitution of glycine by arginine at codon 388 (G388 to R388), have shown a correlation between FGFR4 R388 and aggressive disease progression in cancer patients. Here, we studied the differential effects of the two FGFR4 isotypes on cellular signalling and motility in the MDA-MB-231 human breast cancer cell model. cDNA array analysis showed the ability of FGFR4 G388 to suppress expression of specific genes involved in invasiveness and motility. Further investigations concentrating on cell signalling and motility revealed an abrogation of phosphatidylinositol-3-kinase-dependent LPA-induced Akt activation and cell migration due to downregulation of the LPA receptor Edg-2 in FGFR4 G388-expressing MDA-MB-231 cells. Moreover, FGFR4 G388 expression attenuated the invasivity of the breast cancer cell line and decreased small Rho GTPase activity. We conclude that FGFR4 G388 suppresses cell motility of invasive breast cancer cells by altering signalling pathways and the expression of genes that are required for metastasis. Therefore, the positive effect of FGFR4 R388 on disease progression appears to result from a loss of the tumour suppressor activity displayed by FGFR4 G388 rather than the acquisition or enhancement of oncogenic potential.     

Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrested human MCF-7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC-induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF-7 cells cultivated in medium with and without phenol red. The kinetics of MCF-7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF-7 cells cultivated in phenol red-deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti-proliferative and pro-apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC-induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER-positive breast cancers by CDK inhibitors is diminished in the presence of estrogen-mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors.     

Endophytic bacterial diversity in poplar trees growing on a BTEX-contaminated site: the characterisation of isolates with potential to enhance phytoremediation

The diversity of endophytic bacteria found in association with poplar was investigated as part of a larger study to assess the possibility and practicality of using endophytic bacteria to enhance in situ phytoremediation. Endophytic bacteria were isolated from the root, stem and leaf of two cultivars of poplar tree growing on a site contaminated with BTEX compounds. They were further characterised genotypically by comparative sequence analysis of partial 16S rRNA genes and BOX-PCR genomic DNA fingerprinting, and phenotypically by their tolerance to a range of target pollutants, heavy metals and antibiotics. One hundred and 21 stable, morphologically distinct isolates were obtained, belonging to 21 genera, although six isolates could not be identified with confidence to a genus. The endophytic bacteria exhibited marked spatial compartmentalisation within the plant, suggesting there are likely to be species-specific and non-specific associations between bacteria and plants. A number of isolates demonstrated the ability to degrade BTEX compounds or to grow in the presence of TCE. This study demonstrates that within the diverse bacterial communities found in poplar several endophytic strains are present that have the potential to enhance phytoremediation strategies.