Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs

Background  

By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.     

Gene expression profiling of breast carcinomas using nylon DNA arrays

Clinically very heterogeneous, breast cancer prognosis and treatment response are difficult to predict with the current prognostic histoclinical parameters. Mammary oncogenesis remains poorly understood. DNA array technology allows the simultaneous analysis of the mRNA expression levels of thousands of genes in biological samples. Applied to breast tumours, expression profiles will boost our knowledge of oncogenesis, will offer new potential therapeutic targets and new prognostic and predictive markers. Today, the most accessible approach for academic research teams is that of Nylon DNA arrays with radioactive detection, which in addition allows profiling of small clinical samples.     

Fluorescence in situ hybridization (CARD-FISH) of microorganisms in hydrocarbon contaminated aquifer sediment samples

Groundwater ecosystems are the most important sources of drinking water worldwide but they are threatened by contamination and overexploitation. Petroleum spills account for the most common source of contamination and the high carbon load results in anoxia and steep geochemical gradients. Microbes play a major role in the transformation of petroleum hydrocarbons into less toxic substances. To investigate microbial populations at the single cell level, fluorescence in situ hybridization (FISH) is now a well-established technique. Recently, however, catalyzed reporter deposition (CARD)-FISH has been introduced for the detection of microbes from oligotrophic environments. Nevertheless, petroleum contaminated aquifers present a worst case scenario for FISH techniques due to the combination of high background fluorescence of hydrocarbons and the presence of small microbial cells caused by the low turnover rates characteristic of groundwater ecosystems. It is therefore not surprising that studies of microorganisms from such sites are mostly based on cultivation techniques, fingerprinting, and amplicon sequencing. However, to reveal the population dynamics and interspecies relationships of the key participants of contaminant degradation, FISH is an indispensable tool. In this study, a protocol for FISH was developed in combination with cell quantification using an automated counting microscope. The protocol includes the separation and purification of microbial cells from sediment particles, cell permeabilization and, finally, CARD-FISH in a microwave oven. As a proof of principle, the distribution of Archaea and Bacteria was shown in 60 sediment samples taken across the contaminant plume of an aquifer (Leuna, Germany), which has been heavily contaminated with several ten-thousand tonnes of petroleum hydrocarbons since World War II.     

Neuropeptide Y-induced phase shifts of PER2::LUC rhythms are mediated by long-term suppression of neuronal excitability in a phase-specific manner

Endogenous circadian rhythms are entrained to the 24-h light/dark cycle by both light and nonphotic stimuli. During the day, nonphotic stimuli, such as novel wheel-induced exercise, produce large phase advances. Neuropeptide Y (NPY) release from the thalamus onto suprachiasmatic nucleus (SCN) neurons at least partially mediates this nonphotic signal. The authors examined the hypothesis that NPY-induced phase advances are accompanied by suppression of PER2 and are mediated by long-term depression of neuronal excitability in a phase-specific manner. First, it was found that NPY-induced phase advances in PER2::LUC SCN cultures are largest when NPY (2.35 μM) is given in the early part of the day (circadian time [CT] 0-6). In addition, PER2::LUC levels in NPY-treated (compared to vehicle-treated) samples were suppressed beginning 6-7?h after treatment. Similar NPY application to organotypic Per1::GFP SCN cultures resulted in long-term suppression of spike rate of green fluorescent protein-positive (GFP+) cells when slices were treated with NPY during the early or middle of the day (zeitgeber time [ZT] 2 or 6), but not during the late day (ZT 10). Furthermore, 1-h bath application of NPY to acute SCN brain slices decreased general neuronal activity measured through extracellular recordings. Finally, NPY-induced phase advances of PER2::LUC rhythms were blocked by latent depolarization with 34.5?mM K(+) 3?h after NPY application. These results suggest that NPY-induced phase advances may be mediated by long-term depression of neuronal excitability. This model is consistent with findings in other brain regions that NPY-induced persistent hyperpolarization underlies mechanisms of energy homeostasis, anxiety-related behavior, and thalamocortical synchronous firing.     

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection

HIV-1 mutations, which reduce or abolish CTL responses against virus-infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV-1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele-associated substitutions of amino acids within or near CD8 T-cell epitopes. In these cases, the non-adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population-based studies have independently identified HLA-associated viral changes, which lead to the formation of a new T-cell epitope, suggesting that the immune responses that these variants or 'neo-epitopes' elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T-cell responses that result from viral adaptation in 125 HLA-genotyped individuals with chronic HIV-1 infection. Neo-epitopes included well-characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T-cell responses against the neo-epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non-adapted epitopes, but were not more cytotoxic. Neo-epitope formation and emergence of cognate T-cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV-1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non-protective T-cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV-1 sequences.     

Nitrogen dioxide oxidizes mitochondrial cytochrome c

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9–19). However, the subsequent re-evaluation of the redox potential of the NO/NO^- couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO₂^? a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3 × 10⁶ M^-1 s^-1 for the reaction of NO₂^? with ferrocytochrome c. The importance of NO₂^? was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO₂^? into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO^- or HNO) or of peroxynitrite.     

Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.     

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

We recently reported that a DNA catalyst (deoxyribozyme) can site-specifically hydrolyze DNA on the minutes time scale. Sequence specificity is provided by Watson-Crick base pairing between the DNA substrate and two oligonucleotide binding arms that flank the 40-nt catalytic region of the deoxyribozyme. The DNA catalyst from our recent in vitro selection effort, 10MD5, can cleave a single-stranded DNA substrate sequence with the aid of Zn(2+) and Mn(2+) cofactors, as long as the substrate cleavage site encompasses the four particular nucleotides ATG^T. Thus, 10MD5 can cleave only 1 out of every 256 (4(4)) arbitrarily chosen DNA sites, which is rather poor substrate sequence tolerance. In this study, we demonstrated substantially broader generality of deoxyribozymes for site-specific DNA hydrolysis. New selection experiments were performed, revealing the optimality of presenting only one or two unpaired DNA substrate nucleotides to the N(40) DNA catalytic region. Comprehensive selections were then performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotide identities at the cleavage site (e.g. T^G), while retaining Watson-Crick sequence generality beyond those nucleotides along with useful cleavage rates. These findings establish experimentally that broadly sequence-tolerant and site-specific deoxyribozymes are readily identified for hydrolysis of single-stranded DNA.     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

The physiological arms race: Exploring thermal acclimation among interacting species

Thermal acclimation is often expected to increase performance during survival-related interactions, such as prey-capture and predator escape. However, few studies have examined acclimation responses in the context of the organism's ecology: namely, considering interactions among different species. In this study, we investigated the acclimation responses of three species of aquatic organisms from the same environment that simultaneously interact across different seasons. We predicted that the crimson spotted rainbowfish (Melanotaenia duboulayi) and the freshwater shrimp (Paratya australiensis), which are involved in a predator–prey relationship, would exhibit similar thermal acclimation responses due to an arms race in physiological responses. In contrast, we expected that the backswimmer (Enithares sp.) species from the same environment, which is not commonly preyed upon due to their hard chitinous exterior, would display a limited acclimation response. We found that acute increases in temperature resulted in improved locomotor performance for all three species and an improved prey capture performance for M. duboulayi. Acclimation to 15 °C or 25 °C for 6 weeks did not affect the prey-capture performance of M. duboulayi or the locomotor performance of either M. duboulayi or Enithares sp. However, acclimation to cool temperatures improved the locomotor performance of P. australiensis at both cool and warm temperatures. Thus, the interaction between M. duboulayi (predator) and P. australiensis (prey) is likely to change across seasons via differences in acclimation responses, which could directly affect their behavioural strategies and population dynamics.