Fluorescent labeling of tRNA dihydrouridine residues: Mechanism and distribution

Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH(4), their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNA(Phe) and E. coli tRNA(Arg). The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH(4) reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.     

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.     

New Insights to Self‐Aggregation in Ionic Liquid Electrolytes for High‐Energy Electrochemical Devices

Some cations of ionic liquids (ILs) of interest for high‐energy electrochemical storage devices, such as lithium batteries and supercapacitors, have a structure similar to that of surfactants. For such, it is very important to understand if these IL cations tend to aggregate like surfactants since this would affect the ion mobility and thus the ionic conductivity. The aggregation behaviour of ILs consisting of the bis(trifluoromethanesulfonyl)imide anion and different N‐alkyl‐N‐methyl‐pyrrolidinium cations, with the alkyl chain varied from C₃H₇ to C₈H₁₇, was extensively studied with NMR and Raman methods, also in the presence of Li⁺ cations. ²H NMR spin‐lattice and spin‐spin relaxation rates were analyzed by applying the “two step” model of surfactant dynamics. Here we show that, indeed, the cations in these ILs tend to form aggregates surrounded by the anions. The effect is even more pronounced in the presence of dissolved lithium cations.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing

Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.     

Hepatocyte growth factor-induced ectodomain shedding of cell adhesion molecule L1: role of the L1 cytoplasmic domain

The L1 cell adhesion molecule and its soluble form are tumor-associated proteins and potential markers for tumor staging as well as targets for therapeutic intervention. Soluble L1 is produced by metalloprotease-mediated ectodomain shedding of L1. We investigated effects of hepatocyte growth factor (HGF), a growth factor shown to increase invasiveness of renal carcinoma cells, on ectodomain shedding of L1 from these cells. All of the tested L1-positive renal carcinoma cell lines released a 180-kDa form of L1 into the medium. In the presence of serum, addition of HGF led to a dose-dependent increase in L1 shedding with a maximum reached at 5 ng/ml. In contrast, L1 shedding was inhibited by glial cell line-derived neurotrophic factor (GDNF). The tyrosine kinase inhibitor Genistein reduced basal and HGF-stimulated L1 shedding, indicating that protein phosphorylation is involved. To investigate the role of the L1 intracellular domain, two mutants of the L1 cytoplasmic part were constructed. L1trun lacking the complete intracellular domain showed enhanced basal shedding. In a L1YH mutant, containing the mutation tyrosine 1229 to histidine that deletes the ankyrin binding motif of L1, basal shedding was reduced. Disruption of actin assembly by cytochalasin D also reduced shedding of L1. These results indicate that the cytoplasmic domain regulates basal shedding of L1, and association with the cytoskeleton through the L1 ankyrin binding site is involved. HGF stimulated L1 shedding in both mutants, indicating that receptor-mediated phosphorylation in the L1 cytoplasmic domain is not required for HGF-stimulated shedding.