Discovery of a mRNA Mitochondrial Localization Element in Saccharomyces Cerevisiae by Nonhomologous Random Recombination and In Vivo Selection

In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3’ UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3’ end of various reporter mRNAs to induce mitochondrial localization. Sitedirected mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.Chemistry and Chemical Biolog

Transcription of cis-antisense small RNA MtlS in Vibrio cholerae is regulated by transcription of its target gene mtlA

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA’s target gene

Oral diabetes medication monotherapy and short-term mortality in individuals with type 2 diabetes and coronary artery disease

Objective To determine whether sulfonylurea use, compared with non-sulfonylurea oral diabetes medication use, was associated with 2-year mortality in individuals with well-controlled diabetes and coronary artery disease (CAD). Research design and methods We studied 5352 US veterans with type 2 diabetes, obstructive CAD on coronary angiography, hemoglobin A1c ≤7.5% at the time of catheterization, and taking zero or one oral diabetes medication (categorized as no medications, non-sulfonylurea medication, or sulfonylurea). We estimated the association between medication category and 2-year mortality using inverse probability of treatment-weighted (IPW) standardized mortality differences and IPW multivariable Cox proportional hazards regression. Results 49%, 35%, and 16% of the participants were on no diabetes medications, non-sulfonylurea medications, and sulfonylureas, respectively. In individuals on no medications, non-sulfonylurea medications, and sulfonylureas, the unadjusted mortality rates were 6.6%, 5.2%, and 11.9%, respectively, and the IPW-standardized mortality rates were 5.9%, 6.5%, and 9.7%, respectively. The standardized absolute 2-year mortality difference between non-sulfonylurea and sulfonylurea groups was 3.2% (95% CI 0.7 to 5.7) (p=0.01). In Cox proportional hazards models, the point estimate suggested that sulfonylurea use might be associated with greater hazard of mortality than non-sulfonylurea medication use, but this finding was not statistically significant (HR 1.38 (95% CI 1.00 to 1.93), p=0.05). We did not observe significant mortality differences between individuals on no diabetes medications and non-sulfonylurea users. Conclusions Sulfonylurea use was common (nearly one-third of those taking medications) and was associated with increased 2-year mortality in individuals with obstructive CAD. The significance of the association between sulfonylurea use and mortality was attenuated in fully adjusted survival models. Caution with sulfonylurea use may be warranted for patients with well-controlled diabetes and CAD, and metformin or newer diabetes medications with cardiovascular safety data could be considered as alternatives when individualizing therapy

Complete replication of hepatitis C virus in cell culture.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals

Transcription of cis-antisense small RNA MtlS in Vibrio cholerae is regulated by transcription of its target gene mtlA

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA’s target gene

Zyklophin, a short-acting kappa opioid antagonist, induces scratching in mice

It has been shown previously that norbinaltorphimine (norBNI) and 5΄-guanidinonaltrindole (5΄-GNTI), long-acting kappa opioid receptor (KOPR) antagonists, cause frenzied scratching in mice [1;2]. In the current study, we examined if zyklophin, a short-acting cyclic peptide KOPR antagonist, also elicited scratching behavior. When injected s.c. in the nape of the neck of male Swiss-Webster mice, zyklophin at doses of 0.1, 0.3 and 1 mg/kg induced dose-related hindleg scratching of the neck between 3 and 15 min after injection. Pretreating mice with norBNI (20 mg/kg, i.p.) at 18–20 hr before challenge with zyklophin (0.3 mg/kg) did not markedly affect scratching. Additionally, KOPR −/− mice given 0.3 mg/kg of zyklophin displayed similar levels of scratching as wild-type animals. The absence of KOPR in KOPR −/− mice was confirmed with ex vivo radioligand binding using [3H]U69,593. Taken together, our data suggest that the presence of kappa receptors is not required for the excessive scratching caused by zyklophin. Thus, zyklophin, similar to the structurally different KOPR antagonist 5΄-GNTI, appears to act at other targets to elicit scratching and potentially the sensation of itch

Comparison of boreal ecosystem model sensitivity to variability in climate and forest site parameters

Ecosystem models are useful tools for evaluating environmental controls on carbon and water cycles under past or future conditions. In this paper we compare annual carbon and water fluxes from nine boreal spruce forest ecosystem models in a series of sensitivity simulations. For each comparison, a single climate driver or forest site parameter was altered in a separate sensitivity run. Driver and parameter changes were prescribed principally to be large enough to identify and isolate any major differences in model responses, while also remaining within the range of variability that the boreal forest biome may be exposed to over a time period of several decades. The models simulated plant production, autotrophic and heterotrophic respiration, and evapotranspiration (ET) for a black spruce site in the boreal forest of central Canada (56°N). Results revealed that there were common model responses in gross primary production, plant respiration, and ET fluxes to prescribed changes in air temperature or surface irradiance and to decreased precipitation amounts. The models were also similar in their responses to variations in canopy leaf area, leaf nitrogen content, and surface organic layer thickness. The models had different sensitivities to certain parameters, namely the net primary production response to increased CO2 levels, and the response of soil microbial respiration to precipitation inputs and soil wetness. These differences can be explained by the type (or absence) of photosynthesis-CO2 response curves in the models and by response algorithms of litter and humus decomposition to drying effects in organic soils of the boreal spruce ecosystem. Differences in the couplings of photosynthesis and soil respiration to nitrogen availability may also explain divergent model responses. Sensitivity comparisons imply that past conditions of the ecosystem represented in the models\u27 initial standing wood and soil carbon pools, including historical climate patterns and the time since the last major disturbance, can be as important as potential climatic changes to prediction of the annual ecosystem carbon balance in this boreal spruce forest

Applications and limitations of electron correlation microscopy to study relaxation dynamics in supercooled liquids

Electron correlation microscopy (ECM) is a way to measure structural relaxation times, τ, of liquids with nanometer-scale spatial resolution using coherent electron scattering equivalent of photon correlation spectroscopy. We have applied ECM with a 3.5 nm diameter probe to Pt57.5Cu14.7Ni5.3P22.5 amorphous nanorods and Pd40Ni40P20 bulk metallic glass (BMG) heated inside the STEM into the supercooled liquid region. These data demonstrate that the ECM technique is limited by the characteristics of the time series, which must be at least 40τ to obtain a well-converged correlation function g2(t), and the time per frame, which must be less than 0.1τ to obtain sufficient sampling. A high-speed direct electron camera enables fast acquisition and affords reliable g2(t) data even with low signal per frame

Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kinase, and we propose that this mechanism forms a feedback loop that results in the restoration of basal cAMP levels. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserve