Extracellular DNA in aquatic ecosystems may in part be due to phycovirus activity

In a eutrophic lake, a crash of the algal population was followed by a significant increase in the number of virus-like particles (from ca. 1 10⁶ ml^–1 to ca. 26 10⁶ ml^–1), and soon thereafter by an increase of the amount of extracellular DNA (from ca. 20 µg l^–1 to ca. 40 µg l^–1). The same pattern of correlation between decrease of algae and increase of viruses and extracellular DNA could be demonstrated by an in vitro experiment with a Chlorella-virus-system. Lysis of algae by viruses increased both the number of viruses and the amount of DNA in the culture medium. Extracellular DNA mainly consisted of material with a molecular weight below 500 bp.The Chlorella-virus-system is discussed. It could be used as a model-system for studying the dynamics of interaction of viruses and algae in aquatic ecosystems.     

Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2’,3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)—A Novel and Non-Cytotoxic HO-1 Inducer

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO₂-C₆H₄, Ph, p-OMe-C₆H₄, NO₂, CF₃, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C₆H₄-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C₆H₄-TMC displayed no significant activity. Also, E-α-p-OMe-C₆H₄-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C₆H₄-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C₆H₄-TMC treatment. Overall, E-α-p-OMe-C₆H₄-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.     

The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis. However, the mechanisms controlling such events are not fully understood. We have developed markers that provide the single cell resolution necessary to identify the three modes of division occurring in a developing nervous system: self-expanding, self-renewing, and self-consuming. Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5. Functional in vivo experiments showed that the premature neuronal differentiation and changes in cell cycle parameters caused by SMAD1/5 inhibition were preceded by a reduction of self-expanding divisions in favor of self-consuming divisions. Conversely, SMAD1/5 gain of function promoted self-expanding divisions. Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.     

An assessment of the role of domain F and pest sequences in estrogen receptor half-life and bioactivity

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3–4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [³⁵S]methionine/[³⁵]Scysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.