Snf1 kinases with different beta-subunit isoforms play distinct roles in regulating haploid invasive growth

The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different beta-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.     

Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction,and modulates siRNA activity and allele specificity

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (ⁱE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for ⁱE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the ⁱE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of ⁱE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington''s disease as an example, ⁱE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the ⁱE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.     

Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.     

Transcriptional regulation restricting bone sialoprotein gene expression to both hypertrophic chondrocytes and osteoblasts

This study identifies a cis-acting element that confers tissue-restricted expression to the bone sialoprotein (BSP) gene. Using both gain of function and loss-of function studies, we demonstrate that this element acts as a tissue specific enhancer of BSP expression in osteoblasts and hypertrophic chondrocytes but does not function in non-hypertrophic chondrocytes or fibroblasts. Furthermore, our data demonstrate that binding of this element occurs in correlation with active BSP expression. While Dlx5 has been implicated as the tissue-specific regulator of BSP expression through direct DNA binding at an element with homology to the one under study here, our results demonstrate that Dlx5 does not act as a positive regulator of BSP expression. Finally, mutational analyses of this element demonstrate that while there is homology to putative homeodomain binding elements, this site is unlikely to bind homeodomain factors including Dlx5. Thus, these studies identify an important cis-acting element in the BSP promoter that acts as a tissue-specific enhancer of BSP expression in both osteoblasts and hypertrophic chondrocytes. As such this is the first demonstration of a common regulatory mechanism utilized by both chondrocytes and osteoblasts for the tissue-restricted expression of the BSP gene.     

COMPARATIVE STUDIES ON RESPIRATION : V. THE EFFECT OF ETHER ON THE PRODUCTION OF CARBON DIOXIDE BY ANIMALS.

1. The experiments on frog tadpoles show that with 0.15, 0.37, and 0.55 per cent ether solutions there is a decrease in CO₂ output. The effect is reversible. With these concentrations the breathing movements and body movements remained normal during the experiment. In 3.65 and 7.3 per cent ether there is a decrease of respiration followed by an increase which in turn is followed by a decrease. The increase may reach about three times the normal rate. The increase in the CO₂ output is accompanied by the peeling of the skin. The effect is irreversible. 2. Experiments on an aquatic insect, Dineutes assimilis Aube, show that in 7.3 per cent ether there is a decrease followed by an increase which in turn is followed by a decrease. There is no apparent disintegration of structures in the organism accompanying the increase. The effect is irreversible. 3. The experiments on frog eggs with 7.3 per cent ether show a result similar to that found in aquatic insects. 4. Experiments on Fundulus embryos show that with 0.73 per cent ether there is a reversible decrease in the rate of CO₂ production. In 3.65 per cent ether there is a temporary decrease followed by an increase, after which the rate begins to fall off. In 7.3 per cent ether there is an immediate increase amounting to 307 per cent which is followed by a decrease. The increase in the 3.65 and 7.3 per cent ether is accompanied by irreversible changes leading to death. The decrease found in 0.73 per cent ether is not sufficient to cause narcosis, as is shown by experiments on which the same decrease is produced by lowering the temperature. 5. These experiments show that narcosis is not due to asphyxia. The action of anesthetics is due to some other cause than the effect on respiration. There is a difference between the animals studied and the plants described in this series of articles, since in animals the increase in the CO₂ output is accompanied by irreversible changes leading to death, while this is not necessarily the case in plants. The reversible (narcotic) action of ether on the animals studied was accompanied by a decrease in the carbon dioxide output; in plants this is not ordinarily the case. These facts are of considerable interest, but their interpretation must be left to future investigation.     

A mutation in CLOCK leads to altered dopamine receptor function

Mice with a mutation in the Clock gene (ClockΔ19) have a number of behavioral phenotypes that suggest alterations in dopaminergic transmission. These include hyperactivity, increased exploratory behavior, and increased reward value for drugs of abuse. However, the complex changes in dopaminergic transmission that underlie the behavioral abnormalities in these mice remain unclear. Here we find that a loss of CLOCK function increases dopamine release and turnover in striatum as indicated by increased levels of metabolites HVA and DOPAC, and enhances sensitivity to dopamine receptor antagonists. Interestingly, this enlarged dopaminergic tone results in downstream changes in dopamine receptor (DR) levels with a surprising augmentation of both D1‐ and D2‐type DR protein, but a significant shift in the ratio of D1 : D2 receptors in favor of D2 receptor signaling. These effects have functional consequences for both behavior and intracellular signaling, with alterations in locomotor responses to both D1‐type and D2‐type specific agonists and a blunted response to cAMP activation in the ClockΔ19 mutants. Taken together, these studies further elucidate the abnormalities in dopaminergic transmission that underlie mood, activity, and addictive behaviors.     

Fasting Blood Glucose-A Missing Variable for GFR-Estimation in Type 1 Diabetes?

Objective

Estimation of glomerular filtration rate (eGFR) is one of the current clinical methods for identifying risk for diabetic nephropathy in subjects with type 1 diabetes (T1D). Hyperglycemia is known to influence GFR in T1D and variability in blood glucose at the time of eGFR measurement could introduce bias in eGFR. We hypothesized that simultaneously measured blood glucose would influence eGFR in adults with T1D.

Methods

Longitudinal multivariable mixed-models were employed to investigate the relationships between blood glucose and eGFR by CKD-EPI eGFR_{CYSTATIN C} over 6-years in the Coronary Artery Calcification in Type 1 diabetes (CACTI) study. All subjects with T1D and complete data including blood glucose and cystatin C for at least one of the three visits (n = 616, 554, and 521, respectively) were included in the longitudinal analyses.

Results

In mixed-models adjusting for sex, HbA1c, ACEi/ARB, protein and sodium intake positive associations were observed between simultaneous blood glucose and eGFR_{CYSTATIN C} (β±SE:0.14±0.04 per 10 mg/dL of blood glucose, p<0.0001), and hyperfiltration as a dichotomous outcome (OR: 1.04, 95% CI: 1.01–1.07 per 10 mg/dL of blood glucose, p = 0.02).

Conclusions

In our longitudinal data in subjects with T1D, simultaneous blood glucose has an independent positive effect on eGFR_{CYSTATIN C}. The associations between blood glucose and eGFR_{CYSTATIN C} may bias the accurate detection of early diabetic nephropathy, especially in people with longitudinal variability in blood glucose.     

Distinctive Cytokines as Biomarkers Predicting Fatal Outcome of Severe Staphylococcus aureus Bacteremia in Mice

Invasive Staphylococcus aureus infections are frequently associated with bacteraemia. To support clinical decisions on antibiotic therapy, there is an urgent need for reliable markers as predictors of infection outcome. In the present study in mice, bacteraemia was established by intravenous inoculation of a clinical S. aureus isolate at the LD₅₀ inoculum. As potential biomarkers for fatal outcome, blood culture (qualitative and quantitative), serum levels of C-reactive protein (CRP), as well as 31 selected cytokines and chemokines were assessed during the first three days of infection. A positive S. aureus blood culture, the quantitative blood culture, CRP levels, and levels of eight cytokines were indicative for the presence of S. aureus bacteraemia. However, only tumor necrosis factor (TNF) α, interleukin (IL) 1α, and keratinocyte chemoattractant (KC; a functional homologue of human IL-8) were each significantly elevated in eventually non-surviving infected mice versus eventually surviving infected mice. In severe S. aureus bacteraemia in mice, TNF-α, IL-1α, and KC are biomarkers predicting fatal outcome of infection. KC was a biomarker elevated irrespective the progression of infection, which is very interesting regarding clinical application in view of the heterogeneity of patients experiencing bacteraemia in this respect.     

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.