Benzophenones from Guignardia sp. IFB‐E028, an Endophyte on Hopea hainanensis

The first natural S‐containing benzophenone dimer, named guignasulfide ( 3 ), was isolated from the culture of Guignardia sp. IFB‐E028, an endophytic fungus residing in healthy leaves of Hopea hainanensis. Its structure was determined through correlative analyses of its MS, 1D‐ and 2D‐NMR spectroscopic data. Two other known benzophenone derivatives, monomethylsulochrin and rhizoctonic acid ( 1 and 2 , resp.) were also isolated. Guignasulfide ( 3 ) was more active against the human liver cancer cell line HepG2 (IC₅₀ value: 5.2±0.4 μM ) than metabolites 1 and 2 (IC₅₀ values: 63.5±0.6 and 60.2±0.5 μM ); compounds 1 – 3 showed also moderately inhibitory effects on the human bacterial pathogen Helicobacter pylori with MIC values of 28.9±0.1, 60.2±0.4, and 42.9±0.5 μM , respectively.     

Photoreversion of ultraviolet induced apoptosis in Rat Kangaroo cells

Rat kangaroo(Potorous tridactylus) cells efficiently repair 254 nm ultraviolet light (UV) induced cyclobutane pyrlmidine dimers (CPDs) through photoreactivation, leading to an enhancement of survival when cells are exposed to photoreactivation light (PRL) immediately after UV-irradiation. This work presents evidence that at least part of the UV-irradiated cells die through apoptosis, as demonstrated by DNA fragmentation and chromatin condensation. The induction of this kind of cell death can be reversed through photoreactivation immediately after irradiation, indicating that CPDs are essential signals for the initiation of apoptosis by UV-irradiation. Exposure to PRL 24 h after UV-irradiation does not reverse the induction of apoptosis, implying that the cells are committed to die at this time after irradiation. Inhibition of DNA synthesis during this period of time following UV-irradiation, and before exposure to PRL, does not avoid apoptosis. Since similar results were obtained in G_o confluent and G₁/S synchronized cells, the signals for the UV-induced apoptosis do not seem to be related to a specific phase of cell cycle. Nevertheless, by adding 3-aminobenzamide (3AB—an inhibitor of poly(ADP-ribose) polymerase) in the cell medium after UV-irradiation, apoptosis endpoints were partially reversed if cells are exposed to PRL 24 h later. This result strongly indicates that poly(ADP-ribose) is an intermediary signal for UV-induced apoptosis in mammalian cells.     

Dihydrophenanthrene Dimers: Why and Where It Is Possible to Isolate Their Precursors

An interesting class of compounds of natural origin is dihydrophenanthrene dimers, which are characterized by a series of remarkable biological properties. Considering the hypothesis that each dimer is obtained through a biosynthetic mechanism that involves the coupling of the corresponding radicals of the single dihydrophenanthrene unit, we identified 29 dihydrophenanthrenes. Of these dihydrophenanthrenes, 11 were new compounds that could be isolated from 10 different plant species; 11 had already been identified, but not yet isolated in the 17 different plant species from which the corresponding dimers had been isolated; and 7 were known and had been isolated in the same plant sources of the corresponding dimers. A targeted analysis of several natural extracts from specific plant sources would allow the identification of known or new molecules with potential and/or specific biological activities and, in a final analysis, would confirm the relative biosynthetic mechanism.     

Effect of some drugs on excision repair in E. coli cells.

The intercalating dye ethidium bromide (EB), inhibits excision of pyrimidine dimers from UV-irradiated excision-proficient Escherichia coli B/r hcr+ cells. Inhibition is total at a 2.5 - 10(-4) M concentration 120 min after irradiation with a dose of 750 erg/mm2. The viability of irradiated cells diminishes in proportion to the EB concentration. Under wholly analogous conditions of cultivation and irradiation no inhibitory effect of KCN and caffeine (CFF) and only a slight effect of chloramphenicol (CAP) on dimer excision has been observed. The viability of cells is affected by these compounds but it does not appear to depend on the quantity of excised photoproducts. A change in the secondary structure of DNA induced by intercalation of EB appears to be the reason for the depression of excision of UV photoproducts.     

Synthesis and biological assay of new 2’-deoxyuridine dimers containing a 1,2,3-triazole linker. Part I

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2’-deoxyuridine, thymidine and 5-fluoro-2’-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide–alkyne 1,3-dipolar cycloaddition (click reaction), starting from propargylated 2’-deoxyuridine and 5’-azido-nucleoside derivatives. Their cytotoxic activity was tested in five human cancer cell lines: cervical (HeLa), high grade gliomas (U-118?MG, U-87?MG, T98G), liver (HepG2), and normal human fibroblast cell line (MRC-5) using the sulforhodamine B (SRB) assay. The experiment showed that the obtained dimers with a 1,2,3-triazole moiety were very stable compounds, also in the physiological-like media, and had no anticancer activity.     

Integration of superoxide formation and cristae morphology for mitochondrial redox signaling

The mitochondrial network provides the central cell’s energetic and regulatory unit, which besides ATP and metabolite production participates in cellular signaling through regulated reactive oxygen species (ROS) production and various protein/ion fluxes. The inner membrane forms extensive folds, called cristae, i.e. cavities enfolded from and situated perpendicularly to its inner boundary membrane portion, which encompasses an inner cylinder within the outer membrane tubule. Mitochondrial cristae ultramorphology reflects various metabolic, physiological or pathological states. Since the mitochondrion is typically a predominant superoxide source and generated ROS also serve for the creation of information redox signals, we review known relationships between ROS generation within the respiratory chain complexes of cristae and cristae morphology. Notably, it is emphasized that cristae shape is governed by ATP-synthase dimers, MICOS complexes, OPA1 isoforms and the umbrella of their regulation, and also dependent on local protonmotive force (electrical potential component) in cristae. Cristae are also affected by redox-sensitive kinases/phosphatases or p66SHC. ATP-synthase dimers decrease in the inflated intracristal space, diminishing pH and hypothetically having minimal superoxide formation. Matrix-released signaling superoxide/H₂O₂ is predominantly integrated along mitochondrial tubules, whereas the diffusion of intracristal signaling ROS species is controlled by crista junctions, the widening of which enables specific retrograde redox signaling such as during hypoxic cell adaptation. Other physiological cases of H₂O₂ release from the mitochondrion include the modulation of insulin release in pancreatic β-cells, enhancement of insulin signaling in peripheral tissues, signaling by T-cell receptors, retrograde signaling during the cell cycle and cell differentiation, specifically that of adipocytes.     

Immuno-capture of UVDE generated 3’-OH ends at UV photoproducts

A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3’-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3’-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3’-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

Nucleosome positioning,nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.