Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

Radiosensitizing properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols: two modes of action

We examined radiosensitizing properties of two novel platinum complexes (ethylenediamine(L-malato)platinum(II)), Pt1 and bis(1-ethylimidazole(L-malato)platinum(II)), Pt4. Initial double strand break (DSB) level and DSB rejoining were measured, using pulse field gel electrophoresis (PFGE) in human G1 phase lymphocytes subjected to Pt complex treatment alone and in combination with 10Gy of X-rays. Effects of Pt complex pre-treatment followed by X-irradiation were examined on survival (clonogenic ability) and growth (48 h growth tests) in Chinese hamster ovary (CHO-K1), xrs6 and L5178Y (LY) cells (LY-R and LY-S sublines). Cell cycle distributions of CHO cells after drug treatment were determined with the use of flow cytometry. Pt1 slowed down rejoining of X-ray induced DSB. It exerted a more than additive lethal effect on CHO-K1 cells but not on L5178Y cells subjected to combined Pt complex treatment and X-irradiation. In xrs6 cells the effect of combined Pt1+X treatment was additive. We conclude that, as earlier proposed for other Pt complexes, the radiosensitizing effect of Pt1 is connected with converting repairable DNA damage into irrepairable one (mode (i) of action). The requirements for this mode of sensitization are functional DNA repair systems (nucleotide excision repair (NER) and non-homologous end-joining (NHEJ)). Pt4 does not slow down DSB rejoining. It shows a considerable ability to arrest cells in G2 phase. We assume that Pt4 pre-treatment arrests cells in G2 phase and thus sensitizes to X-rays these cells that have a radiosensitive G2 phase (mode (ii) of action).     

Novel indeno[1,2-b]indoloquinones as inhibitors of the human protein kinase CK2 with antiproliferative activity towards a broad panel of cancer cell lines

We previously reported indeno[1,2-b]indoles as a novel class of potent inhibitors of the human protein kinase CK2. In the present study we prepared two novel quinoid derivatives, the indeno[1,2-b]indoloquinones 6b and 6c, and demonstrated inhibition of the human CK2 by the compounds. Furthermore, we showed substantial antiproliferative activity of both compounds towards a broad panel of human cancer cell lines in the low micromolar range. Whereas the earlier indeno[1,2-b]indoles have been shown to be selective for CK2, the indeno[1,2-b]indoloquinones 6b and 6c also inhibited the AMPK activated protein kinase ARK5, potentially contributing to the anti-cancer effects of the compounds. In addition, with compound 6b we found a very potent inhibitor of the leukemia-associated receptor tyrosine kinase FLT3, with an IC(50) of 0.18 μM.     

Response of foundation macrophytes to near‐natural simulated marine heatwaves

Marine heatwaves have been observed worldwide and are expected to increase in both frequency and intensity due to climate change. Such events may cause ecosystem reconfigurations arising from species range contraction or redistribution, with ecological, economic and social implications. Macrophytes such as the brown seaweed Fucus vesiculosus and the seagrass Zostera marina are foundation species in many coastal ecosystems of the temperate northern hemisphere. Hence, their response to extreme events can potentially determine the fate of associated ecosystems. Macrophyte functioning is intimately linked to the maintenance of photosynthesis, growth and reproduction, and resistance against pathogens, epibionts and grazers. We investigated morphological, physiological, pathological and chemical defence responses of western Baltic Sea F. vesiculosus and Z. marina populations to simulated near‐natural marine heatwaves. Along with (a) the control, which constituted no heatwave but natural stochastic temperature variability (0HW), two treatments were applied: (b) two late‐spring heatwaves (June, July) followed by a summer heatwave (August; 3HW) and (c) a summer heatwave only (1HW). The 3HW treatment was applied to test whether preconditioning events can modulate the potential sensitivity to the summer heatwave. Despite the variety of responses measured in both species, only Z. marina growth was impaired by the accumulative heat stress imposed by the 3HW treatment. Photosynthetic rate, however, remained high after the last heatwave indicating potential for recovery. Only epibacterial abundance was significantly affected in F. vesiculosus. Hence both macrophytes, and in particular F. vesiculosus, seem to be fairly tolerant to short‐term marine heatwaves at least at the intensities applied in this experiment (up to 5°C above mean temperature over a period of 9 days). This may partly be due to the fact that F. vesiculosus grows in a highly variable environment, and may have a high phenotypic plasticity.     

Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)piperidine derivatives as histamine H3R ligands

As a continuation of our search for novel histamine H₃ receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2–21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their binding properties at human histamine H₃ receptor (hH₃R). The highest affinities were observed for pentyl derivatives 6–8 (K_i = 8.8–23.4 nM range) and among them piperidine derivative 6 with K_i = 8.8 nM. Structures 6, 7 were also classified as antagonists in cAMP accumulation assay (with EC₅₀ = 157 and 164 nM, respectively). Moreover, new compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Seven compounds (2–4, 9, 11, 12 and 20) showed anticonvulsant activity at maximal electroshock (MES) test in the dose of 30 mg/kg at 0.5 h. In the subcutaneous pentetrazole (scMET) test compound 4 showed protection at 100 and 300 mg/kg dose at mice, however compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies were undertaken, to explain affinity of compounds at hH₃R (taking into the consideration X-ray analysis of compound 18). In order to estimate “drug-likeness” of selected compounds in silico and experimental evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.     

The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.     

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)—5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)—by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography–tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses.     

Nonadditive Effects of Genes in Human Metabolomics

Genome-wide association studies (GWAS) are widely applied to analyze the genetic effects on phenotypes. With the availability of high-throughput technologies for metabolite measurements, GWAS successfully identified loci that affect metabolite concentrations and underlying pathways. In most GWAS, the effect of each SNP on the phenotype is assumed to be additive. Other genetic models such as recessive, dominant, or overdominant were considered only by very few studies. In contrast to this, there are theories that emphasize the relevance of nonadditive effects as a consequence of physiologic mechanisms. This might be especially important for metabolites because these intermediate phenotypes are closer to the underlying pathways than other traits or diseases. In this study we analyzed systematically nonadditive effects on a large panel of serum metabolites and all possible ratios (22,801 total) in a population-based study [Cooperative Health Research in the Region of Augsburg (KORA) F4, N = 1,785]. We applied four different 1-degree-of-freedom (1-df) tests corresponding to an additive, dominant, recessive, and overdominant trait model as well as a genotypic model with two degree-of-freedom (2-df) that allows a more general consideration of genetic effects. Twenty-three loci were found to be genome-wide significantly associated (Bonferroni corrected P ≤ 2.19 × 10⁻¹²) with at least one metabolite or ratio. For five of them, we show the evidence of nonadditive effects. We replicated 17 loci, including 3 loci with nonadditive effects, in an independent study (TwinsUK, N = 846). In conclusion, we found that most genetic effects on metabolite concentrations and ratios were indeed additive, which verifies the practice of using the additive model for analyzing SNP effects on metabolites.     

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.